Pharmacokinetic modulation and compositions for modified Fn3 polypeptides

ABSTRACT

The present disclosure relates to novel vascular endothelial growth factor receptor (VEGFR)-binding polypeptides and methods for using these polypeptides to inhibit biological activities mediated by vascular endothelial growth factors (VEGFs). The present disclosure also provides various improvements relating to single domain binding polypeptides.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/448,171, entitled “Inhibitors of Type 2 Vascular Endothelial GrowthFactor Receptors,” filed Jun. 5, 2006, which is a continuation ofInternational Application PCT/US04/40885, entitled “Inhibitors of Type 2Vascular Endothelial Growth Factor Receptors,” filed Dec. 6, 2004 whichclaims the benefit of U.S. Provisional Application No. 60/527,886,entitled “Inhibitors of Vascular Endothelial Growth Factor Receptors,”filed Dec. 5, 2003. All of the teachings of the above-referencedapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present disclosure relates to novel vascular endothelial growthfactor receptor (VEGFR)-binding polypeptides and methods for using thesepolypeptides to inhibit biological activities mediated by vascularendothelial growth factors (VEGFs).

Angiogenesis is the process by which new blood vessels are formed frompre-existing capillaries or post capillary venules; it is an importantcomponent of many physiological processes including ovulation, embryonicdevelopment, wound repair, and collateral vascular generation in themyocardium. Angiogenesis is also central to a number of pathologicalconditions such as tumor growth and metastasis, diabetic retinopathy,and macular degeneration. In many instances, the process begins with theactivation of existing vascular endothelial cells in response to avariety of cytokines and growth factors. In cancer, tumor releasedcytokines or angiogenic factors stimulate vascular endothelial cells byinteracting with specific cell surface receptors. The activatedendothelial cells secrete enzymes that degrade the basement membrane ofthe vessels, allowing invasion of the endothelial cells into the tumortissue. Once situated, the endothelial cells differentiate to form newvessel offshoots of pre-existing vessels. The new blood vessels providenutrients to the tumor, facilitating further growth, and also provide aroute for metastasis.

To date, numerous angiogenic factors have been identified, including theparticularly potent factor VEGF. VEGF was initially purified from theconditioned media of folliculostellate cells and from a variety of celllines. More recently a number of structural homologs and alternativelyspliced forms of VEGF have been identified. The various forms of VEGFbind as high affinity ligands to a suite of VEGF receptors (VEGFRs).VEGFRs are tyrosine kinase receptors, many of which are importantregulators of angiogenesis. The VEGFR family includes 3 major subtypes:VEGFR-1, VEGFR-2 (also known as Kinase Insert Domain Receptor, “KDR”, inhumans), and VEGFR-3. Among VEGF forms, VEGF-A, VEGF-C and VEGF-D areknown to bind and activate VEGFR-2.

VEGF, acting through its cognate receptors, can function as anendothelial specific mitogen during angiogenesis. In addition, there issubstantial evidence that VEGF and VEGFRs are up-regulated in conditionscharacterized by inappropriate angiogenesis, such as cancer. As aresult, a great deal of research has focused on the identification oftherapeutics that target and inhibit VEGF or VEGFR.

Current therapeutic approaches that target or inhibit VEGF or VEGFRinclude antibodies, peptides, and small molecule kinase inhibitors. Ofthese, antibodies are the most widely used for in vivo recognition andinhibition of ligands and cellular receptors. Highly specific antibodieshave been used to block receptor-ligand interaction, therebyneutralizing the biological activity of the components, and also tospecifically deliver toxic agents to cells expressing the cognatereceptor on its surface. Although effective, antibodies are large,complex molecules that rely on expression in recombinant mammalian cellsfor production. Antibodies also cause a variety of side effects that areoften undesirable, including activation of complement pathways andantibody-directed cellular cytotoxicity. As a result, there remains aneed for effective therapeutics that can specifically inhibit VEGF/VEGFRpathways as a treatment for disorders characterized by inappropriateangiogenesis, such as cancer.

SUMMARY OF THE INVENTION

In part, this disclosure provides novel, single domain polypeptides thatbind to VEGFR-2 receptors, particularly human VEGFR-2 (also known asKDR) and mouse VEGFR-2 (also known as Flk-1). VEGFR-2 binding proteinsdescribed herein may be used, for example, to detect VEGFR-2 in vivo orin vitro. Additionally, certain VEGFR-2 binding proteins describedherein may be used to treat diseases associated with VEGFR-2-mediatedbiological activity. For example, KDR mediates pro-angiogenic effects ofVEGF, and accordingly, certain KDR binding proteins of the disclosuremay be used to inhibit angiogenesis in a human patient. Certain VEGFR-2binding proteins of the disclosure may be used to treat disorders suchas cancers, inflammatory diseases, autoimmune diseases andretinopathies. Many disorders related to the hyperproliferation of cellsof a tissue will include an angiogenic component, and thus it isexpected that certain VEGFR-2 binding proteins described herein can beused to treat such disorders.

A single domain polypeptide described herein will generally be apolypeptide that binds to a target, such as VEGFR-2, and where targetbinding activity situated within a single structural domain, asdifferentiated from, for example, antibodies and single chainantibodies, where antigen binding activity is generally contributed byboth a heavy chain variable domain and a light chain variable domain.The disclosure also provides larger proteins that may comprise singledomain polypeptides that bind to target. For example, a plurality ofsingle domain polypeptides may be connected to create a compositemolecule with increased avidity. Likewise, a single domain polypeptidemay be attached (e.g., as a fusion protein) to any number of otherpolypeptides. In certain aspects a single domain polypeptide maycomprise at least five to seven beta or beta-like strands distributedamong at least two beta sheets, as exemplified by immunoglobulin andimmunoglobulin-like domains. A beta-like strand is a string of aminoacids that participates in the stabilization of a single domainpolypeptide but does not necessarily adopt a beta strand conformation.Whether a beta-like strand participates in the stabilization of theprotein may be assessed by deleting the string or altering the sequenceof the string and analyzing whether protein stability is diminished.Stability may be assessed by, for example, thermal denaturation andrenaturation studies. Preferably, a single domain polypeptide willinclude no more than two beta-like strands. A beta-like strand will notusually adopt an alpha-helical conformation but may adopt a random coilstructure. In the context of an immunoglobulin domain or animmunoglobulin-like domain, a beta-like strand will most often occur atthe position in the structure that would otherwise be occupied by themost N-terminal beta strand or the most C-terminal beta strand. An aminoacid string which, if situated in the interior of a protein sequencewould normally form a beta strand, may, when situated at a positioncloser to an N- or C-terminus, adopt a conformation that is not clearlya beta strand and is referred to herein as a beta-like strand.

In certain embodiments, the disclosure provides single domainpolypeptides that bind to VEGFR-2. Preferably the single domainpolypeptides bind to human KDR, mouse Flk-1, or both. A single domainpolypeptide may comprise between about 80 and about 150 amino acids thathave a structural organization comprising: at least seven beta strandsor beta-like strands distributed between at least two beta sheets, andat least one loop portion connecting two beta strands or beta-likestrands, which loop portion participates in binding to VEGFR-2. In otherwords a loop portion may link two beta strands, two beta-like strands orone beta strand and one beta-like strand. Typically, one or more of theloop portions will participate in VEGFR-2 binding, although it ispossible that one or more of the beta or beta-like strand portions willalso participate in VEGFR-2 binding, particularly those beta orbeta-like strand portions that are situated closest to the loopportions. A single domain polypeptide may comprise a structural unitthat is an immunoglobulin domain or an immunoglobulin-like domain. Asingle domain polypeptide may bind to any part of VEGFR-2, althoughpolypeptides that bind to an extracellular domain of a VEGFR-2 arepreferred. Binding may be assessed in terms of equilibrium constants(e.g., dissociation, K_(D)) and in terms of kinetic constants (e.g., onrate constant, k_(on) and off rate constant, k_(off)). A single domainpolypeptide will typically be selected to bind to VEGFR-2 with a K_(D)of less than 10⁻⁶ M, or less than 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M or less than10⁻⁹M. VEGFR-2 binding polypeptides may compete for binding with one,two or more members of the VEGF family, particularly VEGF-A, VEGF-C andVEGF-D and may inhibit one or more VEGFR-2-mediated biological events,such as proliferation of endothelial cells, permeabilization of bloodvessels and increased motility in endothelial cells. VEGFR-2 bindingpolypeptides may be used for therapeutic purposes as well as for anypurpose involving the detection or binding of VEGFR-2. Polypeptides fortherapeutic use will generally have a K_(D) of less than 5×10⁻⁸M, lessthan 10⁻⁸M or less than 10⁻⁹M, although higher K_(D) values may betolerated where the k_(off) is sufficiently low or the k_(on) issufficiently high. In certain embodiments, a single domain polypeptidethat binds to VEGFR-2 will comprise a consensus VEGFR-2 binding sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3 and SEQ ID NO:4. Preferably, such sequence will be situated in aloop, particularly the FG loop.

In certain embodiments, the single domain polypeptide comprises animmunoglobulin (Ig) variable domain. The Ig variable domain may, forexample, be selected from the group consisting of: a human V_(L) domain,a human V_(H) domain and a camelid V_(HH) domain. One, two, three ormore loops of the Ig variable domain may participate in binding toVEGFR-2, and typically any of the loops known as CDR1, CDR2 or CDR3 willparticipate in VEGFR-2 binding.

In certain embodiments, the single domain polypeptide comprises animmunoglobulin-like domain. One, two, three or more loops of theimmunoglobulin-like domain may participate in binding to VEGFR-2. Apreferred immunoglobulin-like domain is a fibronectin type III (Fn3)domain. Such domain may comprise, in order from N-terminus toC-terminus, a beta or beta-like strand, A; a loop, AB; a beta strand, B;a loop, BC; a beta strand C; a loop CD; a beta strand D; a loop DE; abeta strand F; a loop FG; and a beta or beta-like strand G. See FIG. 22for an example of the structural organization. Optionally, any or all ofloops AB, BC, CD, DE, EF and FG may participate in VEGFR-2 binding,although preferred loops are BC, DE and FG. A preferred Fn3 domain is anFn3 domain derived from human fibronectin, particularly the 10^(th) Fn3domain of fibronectin, referred to as ¹⁰Fn3. It should be noted thatnone of VEGFR-2 binding polypeptides disclosed herein have an amino acidsequence that is identical to native ¹⁰Fn3; the sequence has beenmodified to obtain VEGFR-2 binding proteins, but proteins having thebasic structural features of ¹⁰Fn3, and particularly those retainingrecognizable sequence homology to the native ¹⁰Fn3 are nonethelessreferred to herein as “¹⁰Fn3 polypeptides”. This nomenclature is similarto that found in the antibody field where, for example, a recombinantantibody V_(L) domain generated against a particular target protein maynot be identical to any naturally occurring V_(L) domain but nonethelessthe protein is recognizably a V_(L) protein. A ¹⁰Fn3 polypeptide may beat least 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the human¹⁰Fn3 domain, shown in SEQ ID NO:5. Much of the variability willgenerally occur in one or more of the loops. Each of the beta orbeta-like strands of a ¹⁰Fn3 polypeptide may consist essentially of anamino acid sequence that is at least 80%, 85%, 90%, 95% or 100%identical to the sequence of a corresponding beta or beta-like strand ofSEQ ID NO: 5, provided that such variation does not disrupt thestability of the polypeptide in physiological conditions. A ¹⁰Fn3polypeptide may have a sequence in each of the loops AB, CD, and EF thatconsists essentially of an amino acid sequence that is at least 80%,85%, 90%, 95% or 100% identical to the sequence of a corresponding loopof SEQ ID NO:5. In many instances, any or all of loops BC, DE, and FGwill be poorly conserved relative to SEQ ID NO:5. For example, all ofloops BC, DE, and FG may be less than 20%, 10%, or 0% identical to theircorresponding loops in SEQ ID NO:5.

In certain embodiments, the disclosure provides a non-antibodypolypeptide comprising a domain having an immunoglobulin-like fold thatbinds to VEGFR-2. The non-antibody polypeptide may have a molecularweight of less than 20 kDa, or less than 15 kDa and will generally bederived (by, for example, alteration of the amino acid sequence) from areference, or “scaffold”, protein, such as an Fn3 scaffold. Thenon-antibody polypeptide may bind VEGFR-2 with a K_(D) less than 10⁻⁶M,or less than 10⁻⁷M, less than 5×10⁻⁸M, less than 10⁻⁸M or less than10⁻⁹M. The unaltered reference protein either will not meaningfully bindto VEGFR-2 or will bind with a K_(D) of greater than 10⁻⁶M. Thenon-antibody polypeptide may inhibit VEGF signaling, particularly wherethe non-antibody polypeptide has a K_(D) of less than 5×10⁻⁸M, less than10⁻⁸M or less than 10⁻⁹M, although higher K_(D) values may be toleratedwhere the k_(off) is sufficiently low (e.g., less than 5×10⁻⁴s⁻¹). Theimmunoglobulin-like fold may be a ¹⁰Fn3 polypeptide.

In certain embodiments, the disclosure provides a polypeptide comprisinga single domain having an immunoglobulin fold that binds to VEGFR-2. Thepolypeptide may have a molecular weight of less than 20 kDa, or lessthan 15 kDa and will generally be derived (by, for example, alterationof the amino acid sequence) from a variable domain of an immunoglobulin.The polypeptide may bind VEGFR-2 with a K_(D) less than 10⁻⁶M, or lessthan 10⁻⁷M, less than 5×10⁻⁸M, less than 10⁻⁸M or less than 10⁻⁹M. Thepolypeptide may inhibit VEGF signaling, particularly where thepolypeptide has a K_(D) of less than 5×10⁻⁸M, less than 10⁻⁸M or lessthan 10⁻⁹M, although higher K_(D) values may be tolerated where thek_(off) is sufficiently low or where the k_(on) is sufficiently high. Incertain preferred embodiments, a single domain polypeptide having animmunoglobulin fold derived from an immunoglobulin light chain variabledomain and capable of binding to VEGFR-2 may comprise an amino acidsequence selected from the group consisting of: SEQ ID NOs:241-310.

In certain preferred embodiments, the disclosure provides VEGFR-2binding polypeptides comprising the amino acid sequence of any of SEQ IDNOs:192-194. In the case of a polypeptide comprising the amino acidsequence of SEQ ID NO:194, a PEG moiety or other moiety of interest, maybe covalently bound to the cysteine at position 93. The PEG moiety mayalso be covalently bonded to an amine moiety in the polypeptide. Theamine moiety may be, for example, a primary amine found at theN-terminus of a polypeptide or an amine group present in an amino acid,such as lysine or arginine. In certain embodiments, the PEG moiety isattached at a position on the polypeptide selected from the groupconsisting of: a) the N-terminus; b) between the N-terminus and the mostN-terminal beta strand or beta-like strand; c) a loop positioned on aface of the polypeptide opposite the target-binding site; d) between theC-terminus and the most C-terminal beta strand or beta-like strand; ande) at the C-terminus.

In certain aspects, the disclosure provides short peptide sequences thatmediate VEGFR-2 binding. Such sequences may mediate VEGFR-2 binding inan isolated form or when inserted into a particular protein structure,such as an immunoglobulin or immunoglobulin-like domain. Examples ofsuch sequences include those disclosed as SEQ ID NOs:1-4 and othersequences that are at least 85%, 90%, or 95% identical to SEQ ID NOs:1-4and retain VEGFR-2 binding activity. Accordingly, the disclosureprovides substantially pure polypeptides comprising an amino acidsequence that is at least 85% identical to the sequence of any of SEQ IDNOs:1-4, wherein said polypeptide binds to a VEGFR-2 and competes with aVEGF species for binding to VEGFR-2. Examples of such polypeptidesinclude a polypeptide comprising an amino acid sequence that is at least80%, 85%, 90%, 95% or 100% identical to an amino acid sequence at least85% identical to the sequence of any of SEQ ID NOs:6-183, 186-197, 199and 311-528. Preferably such polypeptide will inhibit a biologicalactivity of VEGF and may bind to VEGFR-2 with a K_(D) less than 10⁻⁶M,or less than 10⁻⁷M, less than 5×10⁻⁸M, less than 10⁻⁸M or less than10⁻⁹M.

In certain embodiments, any of the VEGFR-2 binding polypeptidesdescribed herein may be bound to one or more additional moieties,including, for example, a moiety that also binds to VEGFR-2 (e.g., asecond identical or different VEGFR-2 binding polypeptide), a moietythat binds to a different target (e.g., to create a dual-specificitybinding agent), a labeling moiety, a moiety that facilitates proteinpurification or a moiety that provides improved pharmacokinetics.Improved pharmacokinetics may be assessed according to the perceivedtherapeutic need. Often it is desirable to increase bioavailabilityand/or increase the time between doses, possibly by increasing the timethat a protein remains available in the serum after dosing. In someinstances, it is desirable to improve the continuity of the serumconcentration of the protein over time (e.g., decrease the difference inserum concentration of the protein shortly after administration andshortly before the next administration). Moieties that tend to slowclearance of a protein from the blood include polyethylene glycol,sugars (e.g. sialic acid), and well-tolerated protein moieties (e.g., Fcfragment or serum albumin). The single domain polypeptide may beattached to a moiety that reduces the clearance rate of the polypeptidein a mammal (e.g., mouse, rat, or human) by greater than three-foldrelative to the unmodified polypeptide. Other measures of improvedpharmacokinetics may include serum half-life, which is often dividedinto an alpha phase and a beta phase. Either or both phases may beimproved significantly by addition of an appropriate moiety. Wherepolyethylene glycol is employed, one or more PEG molecules may beattached at different positions in the protein, and such attachment maybe achieved by reaction with amines, thiols or other suitable reactivegroups. Pegylation may be achieved by site-directed pegylation, whereina suitable reactive group is introduced into the protein to create asite where pegylation preferentially occurs. In a preferred embodiment,the protein is modified so as to have a cysteine residue at a desiredposition, permitting site directed pegylation on the cysteine. PEG mayvary widely in molecular weight and may be branched or linear. Notably,the present disclosure establishes that pegylation is compatible withtarget binding activity of ¹⁰Fn3 polypeptides and, further, thatpegylation does improve the pharmacokinetics of such polypeptides.Accordingly, in one embodiment, the disclosure provides pegylated formsof ¹⁰Fn3 polypeptides, regardless of the target that can be bound bysuch polypeptides.

In certain embodiments, the disclosure provides a formulation comprisingany of the VEGFR-2 binding polypeptides disclosed herein. A formulationmay be a therapeutic formulation comprising a VEGFR-2 bindingpolypeptide and a pharmaceutically acceptable carrier. A formulation mayalso be a combination formulation, comprising an additional activeagent, such as an anti-cancer agent or an anti-angiogenic agent.

In certain aspects, the disclosure provides methods for using a VEGFR-2binding protein to inhibit a VEGF biological activity in a cell or toinhibit a biological activity mediated by VEGFR-2. The cell may besituated in vivo or ex vivo, and may be, for example, a cell of a livingorganism, a cultured cell or a cell in a tissue sample. The method maycomprise contacting said cell with any of the VEGFR-2-inhibitingpolypeptides disclosed herein, in an amount and for a time sufficient toinhibit such biological activity.

In certain aspects, the disclosure provides methods for treating asubject having a condition which responds to the inhibition of VEGF orVEGFR-2. Such a method may comprise administering to said subject aneffective amount of any of the VEGFR-2 inhibiting polypeptides describedherein. A condition may be one that is characterized by inappropriateangiogenesis. A condition may be a hyperproliferative condition.Examples of conditions (or disorders) suitable for treatment includeautoimmune disorders, inflammatory disorders, retinopathies(particularly proliferative retinopathies), and cancers. Any of theVEGFR-2 inhibiting polypeptides described herein may be used for thepreparation of a medicament for the treatment of a disorder,particularly a disorder selected from the group consisting of: anautoimmune disorder, an inflammatory disorder, a retinopathy, and acancer.

In certain aspects, the disclosure provides methods for detectingVEGFR-2 in a sample. A method may comprise contacting the sample with aVEGFR-2 binding polypeptide described herein, wherein said contacting iscarried out under conditions that allow polypeptide-VEGFR-2 complexformation; and detecting said complex, thereby detecting said VEGFR-2 insaid sample. Detection may be carried out using any technique known inthe art, such as, for example, radiography, immunological assay,fluorescence detection, mass spectroscopy, or surface plasmon resonance.The sample will often by a biological sample, such as a biopsy, andparticularly a biopsy of a tumor, a suspected tumor or a tissuesuspected of undergoing unwanted angiogenesis. The sample may be from ahuman or other mammal. The VEGFR-2 binding polypeptide may be labeledwith a labeling moiety, such as a radioactive moiety, a fluorescentmoiety, a chromogenic moiety, a chemiluminescent moiety, or a haptenmoiety. The VEGFR-2 binding polypeptide may be immobilized on a solidsupport.

Another aspect of the disclosure relates to a nucleic acid comprising anucleic acid sequence encoding a polypeptide disclosed herein. Incertain embodiments, a nucleic acid may comprise a nucleic acid sequenceencoding a polypeptide selected from the group consisting of any of SEQID Nos. 6-183, 186-197, 199 and 241-528. In certain embodiments, anucleic acid comprises a nucleic acid sequence that hybridizes instringent conditions to a nucleic acid sequence of SEQ ID NO:184 andencodes a polypeptide that binds to human KDR with a KD of less than1×10-6M. In particular embodiments, nucleic acid may comprise a nucleicacid sequence selected from the group consisting of SEQ ID NO:184 andSEQ ID NO:185.

A further aspect of the disclosure relates to an expression vectorcomprising a nucleic acid operably linked with a promoter, wherein thenucleic acid encodes a polypeptide disclosed herein. Another aspect ofthe disclosure relates to a cell comprising a nucleic acid disclosedherein. Also provided is a method of producing the polypeptide thatbinds VEGFR-2, e.g., KDR, comprising: expressing a nucleic acid encodinga polypeptide of the disclosure. In certain embodiments, the nucleicacid may comprise a sequence that encodes a polypeptide selected fromthe group consisting of any of SEQ ID Nos. 6-183, 186-197, 199 and241-528. In certain embodiments, the nucleic acid comprises a sequencethat hybridizes in stringent conditions to a nucleic acid sequence ofSEQ ID NO:184. In certain embodiments, the nucleic acid comprises anucleic acid sequence selected from the group consisting of SEQ IDNO:184 and SEQ ID NO:185. In certain embodiments, the nucleic acid isexpressed in a cell. Alternatively, the nucleic acid is expressed in acell-free system.

In certain aspects, the disclosure provides discoveries that may beapplicable to any ¹⁰Fn3 polypeptide, regardless of which target thepolypeptide is engineered to bind. As noted above, the disclosuredemonstrates that PEG can be used successfully to improve thepharmacokinetics of a ¹⁰Fn3 polypeptide, while not interferingmeaningfully with target binding. Accordingly, the disclosure providespegylated ¹⁰Fn3 polypeptides that bind to target and have improvedpharmacokinetics relative to the non-pegylated polypeptide. In a furtherembodiment, the disclosure demonstrates that a deletion of the firsteight amino acids of a ¹⁰Fn3 polypeptide can increase target bindingaffinity. Accordingly, the disclosure provides ¹⁰Fn3 polypeptideslacking the initial eight amino acids (amino acids numbered in referenceto the sequence of SEQ ID No:5). It is understood that one or two aminoacids may be added back to the deleted form of the polypeptide so as tofacilitate translation and proper processing. The disclosuredemonstrates that subcutaneous administration of a ¹⁰Fn3 polypeptideresults in a delayed release of polypeptide into the bloodstream and adecreased maximum serum concentration of the ¹⁰Fn3 polypeptide.Accordingly, the disclosure provides methods for administering a ¹⁰Fn3polypeptide to a patient by a subcutaneous administration. This route ofadministration may be useful to achieve a delayed release relative tointravenous administration, and/or to decrease the maximum serumconcentration of the ¹⁰Fn3 polypeptide by at least 25% or at least 50%relative to the maximum serum concentration achieved by intravenousadministration of an equal dosage. The administered ¹⁰Fn3 polypeptidemay be attached to a moiety that increases the serum half-life (ordecreases clearance rate, or similarly affects another pharmacokineticparameter) of the ¹⁰Fn3 polypeptide, such as a polyethylene glycolmoiety. Preferably, the administered ¹⁰Fn3 polypeptide comprises anamino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%identical to SEQ ID NO:5.

In certain aspects, the disclosure provides single domain polypeptidesthat bind to a preselected target protein from a first mammal and to ahomolog thereof from a second mammal. Such single domain polypeptidesare particularly useful where the first mammal is a human and the secondmammal is a desirable mammal in which to conduct preclinical testing,such as a mouse, rat, guinea pig, dog, or non-human primate. Thedisclosure demonstrates that single domain polypeptides can beengineered to have such dual specificity, and that the dual specificitysimplifies drug development by allowing testing of the same polypeptidein human cells, human subjects and animal models. Preferably, thepreselected target protein of the first mammal and the homolog thereoffrom the second mammal are sufficiently similar in amino acid sequenceto allow generation of dual specificity polypeptides. For example, thepreselected target protein and the homolog from the second mammal mayshare at least 80%, 90%, or 95% identity across a region of at least 50amino acids, and optionally may share at least 80%, 90%, or 95% identityacross the entire protein sequence or across the sequence of theextracellular domain, in the case of a membrane protein. A single domainpolypeptide with this type of dual specificity binding characteristicmay comprise an immunoglobulin or immunoglobulin-like domain, and willpreferably bind to both the preselected human target protein and to thehomolog thereof with a dissociation constant of less than 1×10⁻⁶M,1×10⁻⁷M, 5×10⁻⁸M, 1×10⁻⁸M or 1×10⁻⁹M.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are graphs and images depicting the characterization ofKDR-binding single clones from Round 6 of KDR selection. FIG. 1A is agraph showing the specific binding of fibronectin-based binding proteinsto 25 nM of KDR-Fc analyzed in radioactive equilibrium binding assay.FIG. 1B is a graph showing the inhibition of specific binding of KDR-Fcand selected fibronectin based binding proteins in the presence of100-fold excess of VEGF₁₆₅. As shown in this figure, certain bindingproteins bound KDR-Fc competitively with VEGF₁₆₅ while others,exemplified by clone 8, did not compete with VEGF₁₆₅. FIG. 1C is a graphshowing the inhibition of KDR-Fc interaction with immobilized VEGF₁₆₅ inpresence of selected fibronectin based binding proteins analyzed inBIAcore. FIG. 1D is an image showing binding of VR28 to KDR-expressingand control cells detected by immunofluorescence.

FIG. 2 is a graph showing the selection profile for the affinitymaturation of VR28 KDR binder. Shown at left is binding of the VR28clone to KDR-Fc and Flk1-Fc (very low, unlabeled bar). Shown at centeris binding of a crude mutagenized pool and subsequent enrichment roundsto KDR-Fc. Shown at right is binding of further enrichment rounds toFlk-1-Fc. Binding was estimated in radioactive equilibrium binding assayas a percentage of input, using 1 nM KDR-Fc or Flk1-Fc.

FIGS. 3A and 3B are graphs depicting the characterization of KDR-bindingsingle clones from Round 4 of anti-KDR affinity maturation of VR28binder. FIG. 3A shows the saturation binding of VR28 (-▪-) and affinitymatured K1 (-▴-), K6 (-▾-), K9 (-♦-), K10 (-●-), K12 (-□-), K13 (-Δ-),K14 (-∇-), K15 (-⋄-) to KDR-Fc in radioactive equilibrium binding assayFIG. 3B shows the binding of clones with and without N-terminal deletionto KDR-Fc. Deletion Δ 1-8 in the N-terminus of fibronectin-based bindingproteins improved binding to KDR-Fc. The data represents an averageKDR-Fc binding of 23 independent clones with and without N-terminaldeletion.

FIG. 4 is a graph showing the binding of the selected clones to KDR andFlk-1. Specific binding of VR28 and selected clones after four rounds ofaffinity maturation to human KDR (K clones) and seven rounds of affinitymaturation to human (KDR) and mouse (flk-1) (E clones). VEGFR-2-Fcchimeras were compared in radioactive equilibrium binding assay. Thedata represents an average of 3 independent experiments. As shown here,maturation against both mouse and human VEGFR-2 proteins producesbinders that bind to both proteins.

FIGS. 5A and 5B are graphs showing the characterization ofVEGFR-2-binding single clones from Round 7 of affinity maturation ofVR28 binder. Saturation binding of VR28 (-▪-) and specificity matured E3(-▴-), E5 (-▾-), E6 (-♦-), E9 (-●-), E18 (-□-), E19 (-Δ-), E25 (-∇-),E26 (-⋄-), E28 (-◯-), E29 (-×-) clones to KDR (FIG. 5A) and Flk1 (FIG.5B)-Fc chimeras was tested in radioactive equilibrium binding assay.

FIGS. 6A and 6B are graphs showing the characterization of VEGFR-2binding by single clones from Round 7 of affinity maturation of the VR28binder. FIG. 6A shows the importance of arginine at positions 79 and 82in binders with dual specificity to human and mouse VEGFR-2 for bindingto mouse VEGFR-2 (Flk1). When either of these positions was replaced byamino acid other than R (X79=E, Q, W, P; X82=L, K), binding to Flk1 butnot to KDR significantly decreased. FIG. 6B shows the importance of allthree variable loops (BC, DE and FG) of KDR fibronectin-based bindingproteins for binding to the target in these proteins. Substitution ofeach loop at a time by NNS sequence affected binding to KDR and Flk1.The binding data is an average from E6 and E26 clones.

FIGS. 7A and 7B are graphs showing the binding of selectedfibronectin-based binding proteins to CHO cells expressing human KDRreceptor (FIG. 7A) and EpoR-Flk1 chimera (FIG. 7B). E18 (-▪-), E19(-▴-), E26 (-▾-), E29 (-♦-) and WT (-□-) fibronectin-based scaffoldproteins were tested. No binding to control CHO cells was observed (datanot shown).

FIGS. 8A and 8B are graphs showing the inhibition of VEGF-inducedproliferation of Ba/F3-KDR (FIG. 8A) and Ba/F3-Flk1 (FIG. 8B) cells,expressing KDR and Flk1 in the presence of different amounts offibronectin-based binding proteins: E18 (-▪-), E19 (-▴-), E26 (-▾-), E29(-♦-), M5 (-●-), WT (-□-) and anti-KDR or anti-flk-1 Ab (-Δ-). The datarepresents an average of 2 independent experiments.

FIG. 9 is a graph showing the results of a HUVEC proliferation assay inthe presence of different amounts of fibronectin-based scaffoldproteins: E18 (-▪-), E19 (-▴-), E26 (-▾-), E29 (-♦-), M5 (-●-), WT(-□-). The data represents an average of 2 independent experiments. Asshown, the KDR binding proteins caused a decrease in proliferation byapproximately 40%.

FIG. 10 is a set of graphs showing the reversible refolding of M5FL inoptimized buffer.

FIG. 11 is an image showing SDS-PAGE analysis of pegylated forms ofM5FL. M, molecular weight markers [Sea Blue Plus, Invitrogen]; −, M5FLalone; 20, M5FL with 20 kD PEG; 40, M5FL with 40 kD PEG.

FIG. 12 is a graph showing the inhibition of VEGF-induced proliferationof Ba/F3-KDR cells with differing amounts of M5FL (-♦-), M5FL PEG20(-▪-) and M5FL PEG40 (-▴-), respectively. Pegylation has little or noeffect on M5FL activity in this assay.

FIG. 13 shows western analysis of VEGFR-2 signaling in endothelialcells. Phospho VEGFR-2—Visualization of phosphorylated VEGFR-2.VEGFR-2—Sample loading control. Phospho ERK1/2—Visualization ofphosphorylated ERK1/2 (MAPK). ERK1—Sample loading control. The resultsdemonstrated that 130 pM CT-01 blocks VEGFR-2 activation and signalingby VEGF-A.

FIG. 14 shows that various ¹⁰Fn3-derived molecules (e.g. M5FL, F10,CT-01) can block VEGF-A and VEGF-D signaling.

FIG. 15 shows a comparison of ¹²⁵I native, pegylated CT-01 administeredi.v. & i.p. CT-01 is a 12 kDa protein. It is rapidly cleared from theblood. Addition of a 40 kDa PEG reduces its clearance rate and increasesthe AUC by 10 fold. Half life of 16 hr in rats is equivalent to 2×dosing per week in humans. Administration route: i.p. CT-01-PEG40 has anAUC that is only 50% of an i.v. administration.

FIG. 16 shows the tissue distribution of ¹²⁵I-CT01PEG40 in normal rats.Tissue distribution of ¹²⁵I-CT01PEG40 indicates secretion primarily viathe liver and secondarily via the kidney. This is expected for the highmolecular weight PEG form. No long term accumulation of CT-01PEG40 isdetected.

FIG. 17 is a schematic view of the Miles Assay that measures vascularpermeability.

FIG. 18 shows that CT-01 blocks VEGF in vivo using the Miles Assay. Theresults indicate that 5 mg/kg of CT01-PEG40 blocks VEGF challenge.

FIG. 19 shows that CT-01 inhibits tumor growth using the B16-F10 MurineMelanoma Tumor Assay.

FIG. 20 shows that CT-01 inhibits tumor growth using U87 HumanGlioblastoma.

FIGS. 21A and 21B show the sequences of VEGFR binding polypeptides thatare based on an antibody light chain framework/scaffold (SEQ IDNOs:241-310).

FIG. 22 shows the structural organization for a single domainpolypeptide having an immunoglobulin fold (a V_(H) domain of animmunoglobulin, left side) and a single domain polypeptide having animmunoglobulin-like fold (a ¹⁰Fn3 domain, right side).

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

This specification describes, inter alia, the identification andproduction of novel, single domain polypeptides that bind to VEGFR-2receptors. VEGFR-2, also called KDR in humans and Flk-1 in mice, is theprimary mediator for the pro-angiogenic effects of VEGF signaling.VEGFR-2 is bound and activated by VEGF-A, VEGF-C and VEGF-D. Inendothelial cells, VEGFR-2 activation stimulates cell proliferation andmigration, and in vivo, VEGFR-2 activation triggers angiogenesis andincreases the permeability of the vasculature. Increased angiogenesis iswell-established as an important feature of tumor growth and variousretinopathies, while increased permeability of the vasculature is asignificant event in many inflammatory responses.

The present disclosure provides hundreds of single domain polypeptidesthat bind to VEGFR-2, many of which exhibit in vitro and/or in vivo VEGFantagonist activity. Single domain polypeptides having VEGF antagonistactivity will be useful in numerous therapeutic applications. Anti-KDRantibodies have been established as having in vivo utility againstdiseases and conditions ranging from cancers and complications resultingfrom cancers to proliferative retinopathies, inflammatory disorders andfibrosis. Based on the in vivo and in vitro data presented here, it isexpected that the single domain polypeptides will be useful in treatingthe same spectrum of disorders.

In addition to therapeutic applications, VEGFR-2-binding single domainpolypeptides may be used in any circumstance where it is desirable todetect VEGFR-2. For example, many stem cells express VEGFR-2, includingparticularly useful cells of hematopoietic lineages. KDR-bindingpolypeptides may be used, particularly in a labeled format, to detectstem cells and facilitate cell sorting. In vivo, labeled VEGFR-2-bindingpolypeptides may be used to image tissues in which VEGFR-2 is expressed.Elevated VEGFR-2 expression may be characteristic of tissuesexperiencing particularly high levels of angiogenic or inflammatoryactivity. Histological analyses of tissue samples may also benefit fromdetection of VEGFR-2. For example, it may be desirable to detect VEGFR-2expression in a tumor biopsy in order to assess the likely effectivenessof an anti-VEGFR-2 or anti-VEGF therapy. Interestingly, many of theVEGFR-2 binding proteins disclosed herein bind to VEGFR-2 with nanomolardissociation constants and yet fail to have a significant effect onVEGFR-2 mediated biological events. Accordingly, such binding proteins,may be useful for in vivo visualization techniques or cell-labelingtechniques, where it will often be desirable to selectively label cellsthat express a VEGFR-2 without causing a significant perturbation ofVEGFR-2 mediated events.

This disclosure describes the use of an in vitro display technology,termed PROfusion™, that exploits nucleic acid-protein fusions (RNA- andDNA-protein fusions) to identify novel single domain polypeptides andamino acid motifs that are important for binding to VEGFR-2. Nucleicacid-protein fusion technology is a display technology that covalentlycouples a protein to its encoding genetic information. PROfusion™technology was used to screen collections of nucleic acids encodingsingle domain polypeptides constructed using a scaffold based on thehuman fibronectin type three domain (¹⁰Fn3) or constructed from thevariable domains of antibody light chains. The expressed polypeptides,termed a “library” of scaffold proteins, was screened for polypeptidesthat could bind VEGFR-2 with high affinity. We isolated from thislibrary of scaffold proteins novel single domain polypeptides that bindto VEGFR-2 and that, in some instances, inhibit VEGF biologicalactivities. Furthermore, it was discovered that many independentlyrandomized loops situated in immunoglobulin or immunoglobulin-likescaffolds tended to converge to a related set of consensus sequencesthat participated in VEGFR-2 binding. Therefore, it is expected thatpolypeptides having these consensus sequences will be useful as VEGFR-2binding agents even when separated from the protein context in whichthey were identified. See, for example, SEQ ID Nos. 1-4. Suchpolypeptides may be used as independent, small peptide VEGFR-2 bindingagents or may be situated in other proteins, particularly proteins thatshare an immunoglobulin or immunoglobulin-like fold.

As discussed above, the present disclosure demonstrates that singledomain polypeptides having certain desirable properties, such as highaffinity binding to VEGFR-2, antagonist effects with respect to one ormore of VEGF-A, -C or -D and improved pharmacokinetics, can be used aseffective anti-cancer agents. While it is expected that theeffectiveness of such polypeptides as anti-cancer agents is related tothe role of angiogenesis in cancer, we do not wish to be bound to anyparticular mechanism. It is formally possible that the present singledomain polypeptides are effective against cancers for reasons unrelatedto angiogenic processes.

To our knowledge, the present disclosure represents the first successfuleffort to use an Fn3-based polypeptide to achieve a therapeutic effectin vivo. Many of the improvements and discoveries made in achieving invivo effectiveness will be broadly applicable to other Fn3-basedpolypeptides. In other words, although ligand binding properties of anFn3-based polypeptide will generally be determined by a relatively smallnumber of amino acids situated in solvent accessible loop regions, otherfeatures, such as pharmacokinetic features, of Fn3-based polypeptideswill tend to be determined by the majority of the protein that is notdirectly involved in ligand binding and that is conserved from proteinto protein regardless of the target protein. This has been the case withantibodies, where a few loops, called CDR regions, mediate antigenbinding, while other features of in vivo antibody behavior are largelydictated by the conserved framework regions and constant domains.

By “inhibit” is meant a measurable reduction in a phenomenon, often usedherein in reference to any of the following: the interaction of VEGFwith a VEGFR, VEGF- or VEGFR-mediated angiogenesis, angiogenesis,symptoms of angiogenesis, the viability of VEGFR-containing cells, theviability of VEGF-dependent Ba/F3 cells, or VEGF- or VEGFR-mediatedcellular proliferation as compared to a control sample not treated withthe polypeptide. A polypeptide will inhibit a VEGF- or VEGFR-2 mediatedactivity if the reduction in activity or interaction is at least 10%,preferably 20%, 30%, 40%, or 50%, and more preferably 60%, 70%, 80%, 90%or more.

By “VEGF biological activity” is meant any function of any VEGF familymember acting through any VEGF receptor, but particularly signalingthrough a VEGFR-2 receptor. The VEGF family includes VEGF-A, VEGF-B,VEGF-C, VEGF-D, and placental growth factor (PIGF), as well as variousalternatively spliced forms of VEGF including VEGF121, VEGF145, VEGF165,VEGF189, and VEGF206 (Tischer et al., J. Biol. Chem, 266:11947-11954,1991). The VEGFR family of tyrosine kinase receptors includes VEGFR-1(also known as Flt-1), VEGFR-2 (also known as KDR (human form) or Flk-1(mouse form)), and VEGFR-3 (also known as Flt-4). VEGF ligands bind tothe VEGF receptors to induce, for example, angiogenesis, vasculogenesis,endothelial cell proliferation, vasodilation, and cell migration. VEGFligands can also inhibit apoptosis through binding to their cognatereceptors. VEGFR-2 is believed to be the VEGFR most involved inangiogenesis. A VEGFR-2 or KDR-mediated biological activity is anybiological function in which VEGFR-2 or KDR participates insignificantly, such that antagonism of VEGFR-2 or KDR causes ameasurable decrease in the biological activity. The biological activityof VEGF and VEGFR can be measured by standard assays known in the art.Examples include ligand binding assays and Scatchard plot analysis;receptor dimerization assays; cellular phosphorylation assays; tyrosinekinase phosphorylation assays (see for example Meyer et al., Ann. N.Y.Acad. Sci. 995:200-207, 2003); endothelial cell proliferation assayssuch as BrdU labeling and cell counting experiments; VEGF-dependent cellproliferation assays; and angiogenesis assays. Methods for measuringangiogenesis are standard, and are described, for example, in Jain etal. (Nat. Rev. Cancer 2:266-276, 2002). Angiogenesis can be assayed bymeasuring the number of non-branching blood vessel segments (number ofsegments per unit area), the functional vascular density (total lengthof perfused blood vessel per unit area), the vessel diameter, theformation of vascular channels, or the vessel volume density (total ofcalculated blood vessel volume based on length and diameter of eachsegment per unit area). Exemplary assays for VEGF-mediated proliferationand angiogenesis can be found in U.S. Pat. No. 6,559,126, Lyden et al,Nature Medicine 7:1194 (2001), Jacob et al, Exp. Pathol. 15:1234 (1978)and Bae et al, J. Biol. Chem. 275:13588 (2000). These assays can beperformed using either purified receptor or ligand or both, and can beperformed in vitro or in vivo. These assays can also be performed incells using a genetically introduced or the naturally-occurring ligandor receptor or both. A polypeptide that inhibits the biological activityof VEGF will cause a decrease of at least 10%, preferably 20%, 30%, 40%,or 50%, and more preferably 60%, 70%, 80%, 90% or greater decrease inthe biological activity of VEGF. The inhibition of biological activitycan also be measured by the IC50. Preferably, a polypeptide thatinhibits the biological activity of VEGF or VEGFR-2 will have an IC50 ofless than 100 nM, more preferably less than 10 nM and most preferablyless than 1 nM.

2. Polypeptides

The methodology described herein has been successfully used to developsingle domain VEGFR-2 binding polypeptides derived from two relatedgroups of protein structures: those proteins having an immunoglobulinfold and those proteins having an immunoglobulin-like fold. By a“polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D). Theterm “single domain polypeptide” is used to indicate that the targetbinding activity (e.g., VEGFR-2 binding activity) of the subjectpolypeptide is situated within a single structural domain, asdifferentiated from, for example, antibodies and single chainantibodies, where antigen binding activity is generally contributed byboth a heavy chain variable domain and a light chain variable domain. Itis contemplated that a plurality of single domain polypeptides of thesort disclosed herein could be connected to create a composite moleculewith increased avidity. Likewise, a single domain polypeptide may beattached (e.g., as a fusion protein) to any number of otherpolypeptides, such as fluorescent polypeptides, targeting polypeptidesand polypeptides having a distinct therapeutic effect.

Single domain polypeptides of either the immunoglobulin orimmunoglobulin-like scaffold will tend to share certain structuralfeatures. For example, the polypeptide may comprise between about 80 andabout 150 amino acids, which amino acids are structurally organized intoa set of beta or beta-like strands, forming beta sheets, where the betaor beta-like strands are connected by intervening loop portions.Examples of the structural organization for the heavy chain variabledomain and the ¹⁰Fn3 domain are shown in FIG. 22. The beta sheets formthe stable core of the single domain polypeptides, while creating two“faces” composed of the loops that connect the beta or beta-likestrands. As described herein, these loops can be varied to createcustomized ligand binding sites, and, with proper control, suchvariations can be generated without disrupting the overall stability ofthe protein. In antibodies, three of these loops are the well-knownComplementarity Determining Regions (or “CDRs”).

Scaffolds for formation of a single domain polypeptides should be highlysoluble and stable in physiological conditions. Examples ofimmunoglobulin scaffolds are the single domain V_(H) or V_(L) scaffold,as well as a single domain camelid V_(HH) domain (a form of variableheavy domain found in camelids) or other immunoglobulin variable domainsfound in nature or engineered in the laboratory. In the single domainformat disclosed herein, an immunoglobulin polypeptide need not form adimer with a second polypeptide in order to achieve binding activity.Accordingly, any such polypeptides that naturally contain a cysteinewhich mediates disulfide cross-linking to a second protein can bealtered to eliminate the cysteine. Alternatively, the cysteine may beretained for use in conjugating additional moieties, such as PEG, to thesingle domain polypeptide.

Other scaffolds may be non-antibody scaffold proteins. By “non-antibodyscaffold protein or domain” is meant a non-antibody polypeptide havingan immunoglobulin-like fold. By “immunoglobulin-like fold” is meant aprotein domain of between about 80-150 amino acid residues that includestwo layers of antiparallel beta-sheets, and in which the flat,hydrophobic faces of the two beta-sheets are packed against each other.An example of such a scaffold is the “fibronectin-based scaffoldprotein”, by which is meant a polypeptide based on a fibronectin typeIII domain (Fn3). Fibronectin is a large protein which plays essentialroles in the formation of extracellular matrix and cell-cellinteractions; it consists of many repeats of three types (types I, II,and III) of small domains (Baron et al., 1991). Fn3 itself is theparadigm of a large subfamily which includes portions of cell adhesionmolecules, cell surface hormone and cytokine receptors, chaperoning, andcarbohydrate-binding domains for reviews, see Bork & Doolittle, ProcNatl Acad Sci USA. 1992 Oct. 1; 89(19):8990-4; Bork et al., J Mol Biol.1994 Sep. 30; 242(4):309-20; Campbell & Spitzfaden, Structure. 1994 May15; 2(5):333-7; Harpez & Chothia, J Mol Biol. 1994 May 13;238(4):528-39).

Preferably, the fibronectin-based scaffold protein is a “¹⁰FN3”scaffold, by which is meant a polypeptide variant based on the tenthmodule of the human fibronectin type III protein in which one or more ofthe solvent accessible loops has been randomized or mutated,particularly one or more of the three loops identified as the BC loop(amino acids 23-30), DE loop (amino acids 52-56) and FG loop (aminoacids 77-87) (the numbering scheme is based on the sequence on the tenthType III domain of human fibronectin, with the amino acidsVal-Ser-Asp-Val-Pro representing amino acids numbers 1-5). The aminoacid sequence of the wild-type tenth module of the human fibronectintype III domain is:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITGYAVTGRGDSPASSKPISINYRT (SEQ ID NO:5). Thus, thewild-type BC loop comprises the sequence of DAPAVTVR; the wild-type DEloop comprises the sequence of GSKST; the wild-type FG loop comprisesthe sequence of GRGDSPASSKP. The sequences flanking the BC, DE, and FGloops are also termed Frameworks 1, 2, 3, and 4, e.g., in Tables 1-3.

A variety of improved mutant ¹⁰Fn3 scaffolds have been identified. Amodified Asp7, which is replaced by a non-negatively charged amino acidresidue (e.g., Asn, Lys, etc.). Both of these mutations have the effectof promoting greater stability of the mutant ¹⁰Fn3 at neutral pH ascompared to the wild-type form. A variety of additional alterations inthe ¹⁰Fn3 scaffold that are either beneficial or neutral have beendisclosed. See, for example, Batori et al. Protein Eng. 2002 December;15(12):1015-20; Koide et al., Biochemistry 2001 Aug. 28;40(34):10326-33.

Additionally, several novel modifications to the ¹⁰Fn3 scaffold aredisclosed here. Of particular significance, it was discovered that adeletion of the first 8 amino acids of the wild type human ¹⁰Fn3 led toroughly three-fold improved VEGFR-2 binding. Because the first 8 aminoacids tend to fold into a position that is close to the BC, DE and FGloops, it is expected that this mutation will also improve targetbinding in other ¹⁰Fn3 scaffolds selected for binding to differenttargets. Accordingly, one may construct a library of nucleic acidsencoding ¹⁰Fn3 scaffold that lack the first 8 amino acids and conductscreening in this improved library.

Both the variant and wild-type ¹⁰Fn3 proteins are characterized by thesame structure, namely seven beta-strand domain sequences (designated Athrough and six loop regions (AB loop, BC loop, CD loop, DE loop, EFloop, and FG loop) which connect the seven beta-strand domain sequences.The beta strands positioned closest to the N- and C-termini may adopt abeta-like conformation in solution. In SEQ ID No:5, the AB loopcorresponds to residues 15-16, the BC loop corresponds to residues22-30, the CD loop corresponds to residues 39-45, the DE loopcorresponds to residues 51-55, the EF loop corresponds to residues60-66, and the FG loop corresponds to residues 76-87. As shown in FIG.22, the BC loop, DE loop, and FG loop are all located at the same end ofthe polypeptide. Similarly, immunoglobulin scaffolds tend to have atleast seven beta or beta-like strands, and often nine beta or beta-likestrands.

A single domain polypeptide disclosed herein may have at least five toseven beta or beta-like strands distributed between at least two betasheets, and at least one loop portion connecting two beta or beta-likestrands, which loop portion participates in binding to VEGFR-2,particularly KDR, with the binding characterized by a dissociationconstant that is less than 1×10⁻⁶M, and preferably less than 1×10⁻⁸M. Asdescribed herein, polypeptides having a dissociation constant of lessthan 5×10⁻⁹M are particularly desirable for therapeutic use in vivo toinhibit VEGF signaling. Polypeptides having a dissociation constant ofbetween 1×10⁻⁶M and 5×10⁻⁹M may be desirable for use in detecting orlabeling, ex vivo or in vivo, VEGFR-2 proteins.

Optionally, the VEGFR-2 binding protein will bind specifically toVEGFR-2 relative to other related proteins from the same species. By“specifically binds” is meant a polypeptide that recognizes andinteracts with a target protein (e.g., VEGFR-2) but that does notsubstantially recognize and interact with other molecules in a sample,for example, a biological sample. In preferred embodiments a polypeptideof the invention will specifically bind a VEGFR with a K_(D) at least astight as 500 nM. Preferably, the polypeptide will specifically bind aVEGFR with a K_(D) of 1 pM to 500 nM, more preferably 1 pM to 100 nM,more preferably 1 pM to 10 nM, and most preferably 1 pM to 1 nM orlower.

In general, a library of scaffold single domain polypeptides is screenedto identify specific polypeptide variants that can bind to a chosentarget. These libraries may be, for example, phage display libraries orPROfusion™ libraries.

In an exemplary embodiment, we have exploited a novel in vitroRNA-protein fusion display technology to isolate polypeptides that bindto both human (KDR) and mouse (Flk-1) VEGFR-2 and inhibit VEGF-dependentbiological activities. These polypeptides were identified from librariesof fibronectin-based scaffold proteins (Koide et al, JMB 284:1141(1998)) and libraries of V_(L) domains in which the diversity of CDR3has been increased by swapping with CDR3 domains from a population ofV_(H) molecules. ¹⁰FN3 comprises approximately 94 amino acid residues,as shown in SEQ ID NO:5.

In addition, as described above, amino acid sequences at the N-terminusof ¹⁰Fn3 can also be mutated or deleted. For example, randomization ofthe BC, DE, and FG loops can occur in the context of a full-length 10Fn3or in the context of a ¹⁰Fn3 having a deletion or mutation of 1-8 aminoacids of the N-terminus. For example, the L at position 8 can be mutatedto a Q. After randomization to create a diverse library,fibronectin-based scaffold proteins can be used in a screening assay toselect for polypeptides with a high affinity for a protein, in this casethe VEGFR. (For a detailed description of the RNA-protein fusiontechnology and fibronectin-based scaffold protein library screeningmethods see Szostak et al., U.S. Pat. Nos. 6,258,558; 6,261,804;6,214,553; 6,281,344; 6,207,446; 6,518,018; PCT Publication Numbers WO00/34784; WO 01/64942; WO 02/032925; and Roberts and Szostak, Proc Natl.Acad. Sci. 94:12297-12302, 1997, herein incorporated by reference.)

For the initial selection described herein, three regions of the ¹⁰Fn3at positions 23-29, 52-55 and 77-86 were randomized and used for invitro selection against the extracellular domain of human VEGFR-2 (aminoacids 1-764 of KDR fused to human IgG1Fc). Using mRNA display(RNA-protein fusion) and in vitro selection, we sampled a ¹⁰Fn3-basedlibrary with approximately ten trillion variants. The initial selectionidentified polypeptides with moderate affinity (K_(D)=10-200 nM) thatcompeted with VEGF for binding to KDR (human VEGFR-2). Subsequently, asingle clone (K_(D)=11-13 nM) from the initial selection was subjectedto mutagenesis and further selection. This affinity maturation processyielded new VEGFR binding polypeptides with dissociation constantsbetween 60 pM to 2 nM. KDR binders are shown in Table 3. In addition, wealso isolated polypeptides that could bind to Flk-1, the mouse KDRhomolog, from mutagenized populations of KDR binders that initially hadno detectable binding affinity to Flk-1, resulting in the isolation ofpolypeptides that exhibit dual specificities to both human and mouseVEGFR-2. These polypeptides are shown to bind cells that display KDR orFlk-1 extracellular domains. They also inhibited cell growth in aVEGF-dependent proliferation assay. Polypeptides that bind to KDR andFlk-1 are shown in Table 2, while a selection of preferred KDR bindersand KDR/Flk-1 binders are shown in Table 1.

Using the VEGFR-2 binding polypeptides identified in these selections wedetermined FG loop amino acid consensus sequences required for thebinding of the polypeptides to the VEGFR-2. The sequences are listed asSEQ ID NOs:1-4 below.

VEGFR-2 binding polypeptides, such as those of SEQ ID NOs:1-4, may beformulated alone (as isolated peptides), as part of a ¹⁰Fn3 singledomain polypeptide, as part of a full-length fibronectin, (with afull-length amino terminus or a deleted amino terminus) or a fragmentthereof, in the context of an immunoglobulin (particularly a singledomain immunoglobulin), in the context of another protein having animmunoglobulin-like fold, or in the context of another, unrelatedprotein. The polypeptides can also be formulated as part of a fusionprotein with a heterologous protein that does not itself bind to orcontribute in binding to a VEGFR. In addition, the polypeptides of theinvention can also be fused to nucleic acids. The polypeptides can alsobe engineered as monomers, dimers, or multimers.

Sequences of the Preferred Consensus VEGFR-2 Binding Peptides:

SEQ ID NO:1-(L/M)GXN(G/D)(H/R)EL(L/M)TP

[X can be any amino acid; (/) represents alternative amino acid for thesame position]

SEQ ID NO:2-XERNGRXL(L/M/N)TP

[X can be any amino acid; (/) represents alternative amino acid for thesame position]

SEQ ID NO:3-(D/E)GXNXRXXIP

[X can be any amino acid; (/) represents alternative amino acid for thesame position]

SEQ ID NO:4-(D/E)G(R/P)N(G/E)R(S/L)(S/F)IP

[X can be any amino acid; (/) represents alternative amino acid for thesame position]

Sequences of the Preferred VEGFR-2 Binding ¹⁰Fn3 Polypeptides: SEQ IDNO:6 EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTMGLYGHELLTPISINYRT SEQ ID NO:7EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTDGENGQFLLVPISINYRT SEQ ID NO:8EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTMGPNDNELLTPISINYRT SEQ ID NO:9EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTAGWDDHELFIPISINYRT SEQ ID NO:10EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTSGHNDHMLMIPISINYRT SEQ ID NO:11EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTAGYNDQILMTPISINYRT SEQ ID NO:12EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTFGLYGKELLIPISINYRT SEQ ID NO:13EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTTGPNDRLLFVPISINYRT SEQ ID NO:14EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTDVYNDHEIKTPISINYRT SEQ ID NO:15EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTDGKDGRVLLTPISINYRT SEQ ID NO:16EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTEVHHDREIKTPISINYRT SEQ ID NO:17EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTQAPNDRVLYTPISINYRT SEQ ID NO:18EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTREENDHELLIPISINYRT SEQ ID NO:19EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVTHNGHPLMTPISINYRT SEQ ID NO:20EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTLALKGHELLTPISINYRT SEQ ID NO:21VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVAQNDHELITPISINYRT SEQ ID NO:22VSDVPRDL/QEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPAATISGLKPGVDYTITGYAVTMAQSGHELFTPISINYRT SEQ ID NO:24EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVERNGRVLMTPISINYRT SEQ ID NO:25EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVERNGRHLMTPISINYRT SEQ ID NO:33EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTLERNGRELMTPISINYRT SEQ ID NO:45EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTEERNGRTLRTPISINYRT SEQ ID NO:53EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVERNDRVLFTPISINYRT SEQ ID NO:57EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVERNGRELMTPISINYRT SEQ ID NO:62EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTLERNGRELMVPISINYRT SEQ ID NO:63EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTDGRNDRKLMVPISINYRT SEQ ID NO:68EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTDGQNGRLLNVPISINYRT SEQ ID NO:91EVVAATPTSLLISWRHHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVHWNGRELMTPISINYRT SEQ ID NO:92EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTEEWNGRVLMTPISINYRT SEQ ID NO:93EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVERNGHTLMTPISINYRT SEQ ID NO:94EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVEENGRQLMTPISINYRT SEQ ID NO:95EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTLERNGQVLFTPISINYRT SEQ ID NO:96EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVERNGQVLYTPISINYRT SEQ ID NO:97EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTWGYKDHELLIPISINYRT SEQ ID NO:98EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTLGRNDRELLTPISINYRT SEQ ID NO:99EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTDGPNDRLLNIPISINYRT SEQ ID NO:100EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTFARDGHEILTPISINYRT SEQ ID NO:101EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTLEQNGRELMTPISINYRT SEQ ID NO:102EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTVEENGRVLNTPISINYRT SEQ ID NO:103EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTLEPNGRYLMVPISINYRT SEQ ID NO:104EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITGYAVTEGRNGRELFIPISINYRT SEQ ID NO:154VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPAATISGLKPGVDYTITGYAVTWERNGRELFTPISINYRT SEQ ID NO:156VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPAATISGLKPGVDYTITGYAVTKERNGRELFTPISINYRT SEQ ID NO:172VSDVPRDLEVVAATPTSLLISWRHPHFPTHYYRITYGETGGNSPVQEFTVPLQPPAATISGLKPGVDYTITGYAVTTERTGRELFTPISINYRT SEQ ID NO:173VSDVPRDLEVVAATPTSLLISWRHPHFPTHYYRITYGETGGNSPVQEFTVPLQPPAATISGLKPGVDYTITGYAVTKERSGRELFTPISINYRT SEQ ID NO:175VSDVPRDLEVVAATPTSLLISWRHPHFPTHYYRITYGETGGNSPVQEFTVPLQPPAATISGLKPGVDYTITGYAVTLERDGRELFTPISINYRT SEQ ID NO:177VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPLATISGLKPGVDYTITG/VYAVTKERNGRELFTPISINYRT SEQ ID NO:180VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPTTATISGLKPGVDYTITGYAVTWERNGRELFTPISINYRT SEQ ID NO:181VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPTVATISGLKPGVDYTITGYAVTLERNDRELFTPISINYRT SEQ ID NO:186MGEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPSQ SEQ ID NO:187MGEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPCQ SEQ ID NO:188MVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKP SQ SEQ ID NO:189MGEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGWNGRLLSIPISINYRT SEQ ID NO:190MGEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT SEQ ID NO:191MVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT SEQ ID NO:192 (A core formof the polypeptide referred to herein as CT-01):EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISTNYRT

The CT-01 molecule above has a deletion of the first 8 amino acids andmay include additional amino acids at the N- or C-termini. For example,an additional MG sequence may be placed at the N-terminus. The M willusually be cleaved off, leaving a GEV . . . sequence at the N-terminus.The re-addition of the normal 8 amino acids at the N-terminus alsoproduces a KDR binding protein with desirable properties. The N-terminalmethionine is generally cleaved off to yield a sequence: (SEQ ID NO:193)VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT.

A polypeptide disclosed herein may be modified by one or moreconservative substitutions, particularly in portions of the protein thatare not expected to interact with a target protein. It is expected thatas many as 5%, 10%, 20% or even 30% or more of the amino acids in animmunoglobulin or immunoglobulin-like domain may be altered by aconservative substitution without substantially altering the affinity ofthe protein for target. It may be that such changes will alter theimmunogenicity of the polypeptide in vivo, and where the immunogenicityis decreased, such changes will be desirable. As used herein,“conservative substitutions” are residues that are physically orfunctionally similar to the corresponding reference residues. That is, aconservative substitution and its reference residue have similar size,shape, electric charge, chemical properties including the ability toform covalent or hydrogen bonds, or the like. Preferred conservativesubstitutions are those fulfilling the criteria defined for an acceptedpoint mutation in Dayhoff et al., Atlas of Protein Sequence andStructure 5:345-352 (1978 & Supp.). Examples of conservativesubstitutions are substitutions within the following groups: (a) valine,glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d)aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine,threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,tyrosine.

Polypeptides disclosed herein may also be modified in order to improvepotency, bioavailability, chemical stability, and/or efficacy. Forexample, within one embodiment of the invention D-amino acid peptides,or retroenantio peptide sequences may be generated in order to improvethe bioactivity and chemical stability of a polypeptide structure (see,e.g., Juvvadi et al., J. Am. Chem. Soc. 118: 8989-8997, 1996; Freidingeret al., Science, 210: 656-658, 1980). Lactam constraints (seeFreidinger, supra), and/or azabicycloalkane amino acids as dipeptidesurrogates can also be utilized to improve the biological andpharmacological properties of the native peptides (see, e.g., Hanessianet al., Tetrahedron 53:12789-12854, 1997).

Amide bond surrogates, such as thioamides, secondary and tertiaryamines, heterocycles among others (see review in Spatola, A. F. in“Chemistry and Biochemistry of Amino Acids, Peptides and Proteins”Wenstein, B. Ed. Marcel Dekker, New York, 1983 Vol. 7, pp 267-357) canalso be utilized to prevent enzymatic degradation of the polypeptidebackbone thereby resulting in improved activity. Conversion of linearpolypeptides to cyclic polypeptide analogs can also be utilized toimprove metabolic stability, since cyclic polypeptides are much lesssensitive to enzymatic degradation (see generally, Veber, et al. Nature292:55-58, 1981).

Polypeptides can also be modified utilizing end group capping as estersand amides in order to slow or prevent metabolism and enhancelipophilicity. Dimers of the peptide attached by various linkers mayalso enhance activity and specificity (see for example: Y. Shimohigashiet al, in Peptide Chemistry 1988, Proceedings of the 26th Symposium onPeptide Chemistry, Tokyo, Oct. 24-26, pgs. 47-50, 1989). For additionalexamples of polypeptide modifications, such as non-natural amino acids,see U.S. Pat. No. 6,559,126.

For use in vivo, a form suitable for pegylation may be generated. Forexample, a C-terminal tail comprising a cysteine was added andexpressed, as shown below for a CT-01 form lacking the eight N-terminalamino acids (EIDKPCQ is added at the C-terminus).GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPCQ (SEQ ID NO:194). The pegylatedform of this molecule is used in the in vivo experiments describedbelow. A control form with a serine instead of a cysteine was also used:(SEQ ID NO:195) GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPSQ.

The same C-terminal tails may also be added to CT-01 forms having theN-terminal eight amino acids, such as is shown in SEQ ID NO:193.Additional variants with desirable KDR binding properties were isolated.The following core sequence has a somewhat different FG loop, and may beexpressed with, for example, an N-terminal MG sequence, an N-terminalsequence that restores the 8 deleted amino acids, and/or a C-terminaltail to provide a cysteine for pegylation.EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT (SEQ ID NO:196). Another such variant hasthe core sequence: (SEQ ID NO:197)VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT.

Additionally, preferred single domain immunoglobulin polypeptides in aV_(L) framework were isolated by similar methodology and are disclosedin FIG. 21.

Also included in the present invention are nucleic acid sequencesencoding any of the polypeptides described herein. As appreciated bythose skilled in the art, because of third base degeneracy, almost everyamino acid can be represented by more than one triplet codon in a codingnucleotide sequence. In addition, minor base pair changes may result ina conservative substitution in the amino acid sequence encoded but arenot expected to substantially alter the biological activity of the geneproduct. Therefore, a nucleic acid sequence encoding a polypeptidedescribed herein may be modified slightly in sequence and yet stillencode its respective gene product.

In addition, the polypeptides of the present invention can be used aslead polypeptides that can be further mutated and screened forpolypeptides that bind VEGFR with an even greater affinity. In oneexample, a polypeptide described herein is used as a lead polypeptidewhich is further mutated or randomized to produce polypeptides withamino acid mutations distinct from the lead polypeptide. The furtherrandomized polypeptides can then be used to screen for polypeptides thatinhibit VEGF biological activity as described herein (e.g., bind to aVEGFR and block binding of VEGF to the same receptor).

3. Nucleic Acids and Production of Polypeptides

Polypeptides of the present invention can be produced using any standardmethods known in the art.

In one example, the polypeptides are produced by recombinant DNA methodsby inserting a nucleic acid sequence (e.g., a cDNA) encoding thepolypeptide into a recombinant expression vector and expressing the DNAsequence under conditions promoting expression. Examples of nucleic acidsequences encoding a CT-01 polypeptide disclosed herein are: SEQ IDNO:184 atgggcgaagttgttgctgcgacccccaccagcctactgatcagctggcgccacccgcacttcccgactagatattacaggatcacttacggagaaacaggaggaaatagccctgtccaggagttcactgtgcctctgcagccccccacagctaccatcagcggccttaaacctggagttgattataccatcactgtgtatgctgtcactgacggccggaacgggcgcctcctgagcatcccaatttccattaattaccgcacagaaattgacaaaccatgccag SEQ ID NO:185Atgggcgaagttgttgctgcgacccccaccagcctactgatcagctggcgccacccgcacttcccgactagatattacaggatcacttacggagaaacaggaggaaatagccctgtccaggagttcactgtgcctctgcagccccccacagctaccatcagcggccttaaacctggagttgattataccatcactgtgtatgctgtcactgacggccggaacgggcgcctcctgagcatcccaatttcca ttaattaccgcaca

Nucleic acids encoding any of the various polypeptides disclosed hereinmay be synthesized chemically. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. See for example: Mayfield et al., ProcNatl Acad Sci USA. 2003 Jan. 21; 100(2):438-42; Sinclair et al. ProteinExpr Purif. 2002 October; 26(1):96-105; Connell N D. Curr OpinBiotechnol. 2001 October; 12(5):446-9; Makrides et al. Microbiol Rev.1996 September; 60(3):512-38; and Sharp et al. Yeast. 1991 October;7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants are additionally incorporated.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985),the relevant disclosure of which is hereby incorporated by reference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47,1988). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides disclosed herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

VEGFR-binding polypeptides can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The polypeptide of the present invention can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

4. Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modification include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. See, e.g., Raju et al. Biochemistry. 2001 Jul. 31;40(30):8868-76. Effects of such non-amino acid elements on thefunctionality of a polypeptide may be tested for its antagonizing rolein VEGFR-2 or VEGF function, e.g., its inhibitory effect on angiogenesisor on tumor growth.

In one specific embodiment of the present invention, modified forms ofthe subject soluble polypeptides comprise linking the subject solublepolypeptides to nonproteinaceous polymers. In one specific embodiment,the polymer is polyethylene glycol (“PEG”), polypropylene glycol, orpolyoxyalkylenes, in the manner as set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.Examples of the modified polypeptide of the invention include PEGylatedM5FL and PEGylated CT-01.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula:

X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH (1), where n is 20 to 2300 and X is H or aterminal modification, e.g., a C₁₋₄ alkyl. In one embodiment, the PEG ofthe invention terminates on one end with hydroxy or methoxy, i.e., X isH or CH₃ (“methoxy PEG”). A PEG can contain further chemical groupswhich are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).

Although PEG is well-known, this is, to our knowledge, the firstdemonstration that a pegylated ¹⁰Fn3 polypeptide can be pegylated andretain ligand binding activity. In a preferred embodiment, the pegylated¹⁰Fn3 polypeptide is produced by site-directed pegylation, particularlyby conjugation of PEG to a cysteine moiety at the N- or C-terminus.Accordingly, the present disclosure provides a target-binding ¹⁰Fn3polypeptide with improved pharmacokinetic properties, the polypeptidecomprising: a ¹⁰Fn3 domain having from about 80 to about 150 aminoacids, wherein at least one of the loops of said ¹⁰Fn3 domainparticipate in target binding; and a covalently bound PEG moiety,wherein said ¹⁰Fn3 polypeptide binds to the target with a K_(D) of lessthan 100 nM and has a clearance rate of less than 30 mL/hr/kg in amammal. The PEG moiety may be attached to the ¹⁰Fn3 polypeptide by sitedirected pegylation, such as by attachment to a Cys residue, where theCys residue may be positioned at the N-terminus of the ¹⁰Fn3 polypeptideor between the N-terminus and the most N-terminal beta or beta-likestrand or at the C-terminus of the ¹⁰Fn3 polypeptide or between theC-terminus and the most C-terminal beta or beta-like strand. A Cysresidue may be situated at other positions as well, particularly any ofthe loops that do not participate in target binding. A PEG moiety mayalso be attached by other chemistry, including by conjugation to amines.

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol.Chem., 252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a binding polypeptide containing a PEGmolecule is also known as a conjugated protein, whereas the proteinlacking an attached PEG molecule can be referred to as unconjugated.

A variety of molecular mass forms of PEG can be selected, e.g., fromabout 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to VEGFR-2 binding polypeptides. The number of repeatingunits “n” in the PEG is approximated for the molecular mass described inDaltons. It is preferred that the combined molecular mass of PEG on anactivated linker is suitable for pharmaceutical use. Thus, in oneembodiment, the molecular mass of the PEG molecules does not exceed100,000 Da. For example, if three PEG molecules are attached to alinker, where each PEG molecule has the same molecular mass of 12,000 Da(each n is about 270), then the total molecular mass of PEG on thelinker is about 36,000 Da (total n is about 820). The molecular massesof the PEG attached to the linker can also be different, e.g., of threemolecules on a linker two PEG molecules can be 5,000 Da each (each n isabout 110) and one PEG molecule can be 12,000 Da (n is about 270).

In a specific embodiment of the invention, a VEGFR-2 binding polypeptideis covalently linked to one poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of the binding polypeptide; R being lower alkyl; x being 2 or 3;m being from about 450 to about 950; and n and m being chosen so thatthe molecular weight of the conjugate minus the binding polypeptide isfrom about 10 to 40 kDa. In one embodiment, an binding polypeptide'sε-amino group of a lysine is the available (free) amino group.

The above conjugates may be more specifically presented by formula (II):P—NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR (II), wherein P is the group of abinding polypeptide as described herein, (i.e. without the amino groupor amino groups which form an amide linkage with the carbonyl shown informula (II); and wherein R is lower alkyl; x is 2 or 3; m is from about450 to about 950 and is chosen so that the molecular weight of theconjugate minus the binding polypeptide is from about 10 to about 40kDa. As used herein, the given ranges of “m” have an orientationalmeaning. The ranges of “m” are determined in any case, and exactly, bythe molecular weight of the PEG group.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated binding polypeptide will be usedtherapeutically, the desired dosage, circulation time, resistance toproteolysis, immunogenicity, and other considerations. For a discussionof PEG and its use to enhance the properties of proteins, see N. V.Katre, Advanced Drug Delivery Reviews 10: 91-114 (1993).

In one embodiment of the invention, PEG molecules may be activated toreact with amino groups on a binding polypeptide, such as with lysines(Bencham C. O. et al., Anal. Biochem., 131, 25 (1983); Veronese, F. M.et al., Appl. Biochem., 11, 141 (1985); Zalipsky, S. et al., PolymericDrugs and Drug Delivery Systems, adrs 9-110 ACS Symposium Series 469(1999); Zalipsky, S. et al., Europ. Polym. J., 19, 1177-1183 (1983);Delgado, C. et al., Biotechnology and Applied Biochemistry, 12, 119-128(1990)).

In one specific embodiment, carbonate esters of PEG are used to form thePEG-binding polypeptide conjugates. N,N′-disuccinimidylcarbonate (DSC)may be used in the reaction with PEG to form active mixedPEG-succinimidyl carbonate that may be subsequently reacted with anucleophilic group of a linker or an amino group of a bindingpolypeptide (see U.S. Pat. No. 5,281,698 and U.S. Pat. No. 5,932,462).In a similar type of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

Pegylation of a ¹⁰Fn3 polypeptide can be performed according to themethods of the state of the art, for example by reaction of the bindingpolypeptide with electrophilically active PEGs (supplier: ShearwaterCorp., USA, www.shearwatercorp.com). Preferred PEG reagents of thepresent invention are, e.g., N-hydroxysuccinimidyl propionates(PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C., et al.,Bioconjugate Chem. 6 (1995) 62-69). Such methods may used to pegylatedat an ε-amino group of a binding polypeptide lysine or the N-terminalamino group of the binding polypeptide.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson a binding polypeptide (Sartore, L., et al., Appl. Biochem.Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon. Chem., 7, 363-368(1996); Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat. No.5,766,897). U.S. Pat. Nos. 6,610,281 and 5,766,897 describes exemplaryreactive PEG species that may be coupled to sulfhydryl groups.

In some embodiments where PEG molecules are conjugated to cysteineresidues on a binding polypeptide, the cysteine residues are native tothe binding polypeptide, whereas in other embodiments, one or morecysteine residues are engineered into the binding polypeptide. Mutationsmay be introduced into an binding polypeptide coding sequence togenerate cysteine residues. This might be achieved, for example, bymutating one or more amino acid residues to cysteine. Preferred aminoacids for mutating to a cysteine residue include serine, threonine,alanine and other hydrophilic residues. Preferably, the residue to bemutated to cysteine is a surface-exposed residue. Algorithms arewell-known in the art for predicting surface accessibility of residuesbased on primary sequence or a protein. Alternatively, surface residuesmay be predicted by comparing the amino acid sequences of bindingpolypeptides, given that the crystal structure of the framework based onwhich binding polypeptides are designed and evolved has been solved (seeHimanen et al., Nature. (2001) 20-27; 414(6866):933-8) and thus thesurface-exposed residues identified. In one embodiment, cysteineresidues are introduced into binding polypeptides at or near the N-and/or C-terminus, or within loop regions.

In some embodiments, the pegylated binding polypeptide comprises a PEGmolecule covalently attached to the alpha amino group of the N-terminalamino acid. Site specific N-terminal reductive amination is described inPepinsky et al., (2001) JPET, 297,1059, and U.S. Pat. No. 5,824,784. Theuse of a PEG-aldehyde for the reductive amination of a protein utilizingother available nucleophilic amino groups is described in U.S. Pat. No.4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254, 12579, and inChamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated binding polypeptide comprises one ormore PEG molecules covalently attached to a linker, which in turn isattached to the alpha amino group of the amino acid residue at theN-terminus of the binding polypeptide. Such an approach is disclosed inU.S. Patent Publication No. 2002/0044921 and in WO94/01451.

In one embodiment, a binding polypeptide is pegylated at the C-terminus.In a specific embodiment, a protein is pegylated at the C-terminus bythe introduction of C-terminal azido-methionine and the subsequentconjugation of a methyl-PEG-triarylphosphine compound via the Staudingerreaction. This C-terminal conjugation method is described in Cazalis etal., C-Terminal Site-Specific PEGylation of a Truncated ThrombomodulinMutant with Retention of Full Bioactivity, Bioconjug Chem. 2004;15(5):1005-1009.

Monopegylation of a binding polypeptide can also be produced accordingto the general methods described in WO 94/01451. WO 94/01451 describes amethod for preparing a recombinant polypeptide with a modified terminalamino acid alpha-carbon reactive group. The steps of the method involveforming the recombinant polypeptide and protecting it with one or morebiologically added protecting groups at the N-terminal alpha-amine andC-terminal alpha-carboxyl. The polypeptide can then be reacted withchemical protecting agents to selectively protect reactive side chaingroups and thereby prevent side chain groups from being modified. Thepolypeptide is then cleaved with a cleavage reagent specific for thebiological protecting group to form an unprotected terminal amino acidalpha-carbon reactive group. The unprotected terminal amino acidalpha-carbon reactive group is modified with a chemical modifying agent.The side chain protected terminally modified single copy polypeptide isthen deprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification at the N-and/or C-terminal amino acid of the polypeptide.

The ratio of a binding polypeptide to activated PEG in the conjugationreaction can be from about 1:0.5 to 1:50, between from about 1:1 to1:30, or from about 1:5 to 1:15. Various aqueous buffers can be used inthe present method to catalyze the covalent addition of PEG to thebinding polypeptide. In one embodiment, the pH of a buffer used is fromabout 7.0 to 9.0. In another embodiment, the pH is in a slightly basicrange, e.g., from about 7.5 to 8.5. Buffers having a pKa close toneutral pH range may be used, e.g., phosphate buffer.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated binding polypeptide, such as size exclusion(e.g. gel filtration) and ion exchange chromatography. Products may alsobe separated using SDS-PAGE. Products that may be separated includemono-, di-, tri- poly- and un-pegylated binding polypeptide, as well asfree PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent mono-PEGconjugates represents a good balance of yield and activity. Compositionsin which, for example, at least ninety-two percent or at leastninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment, PEGylated binding polypeptide of the inventioncontain one, two or more PEG moieties. In one embodiment, the PEGmoiety(ies) are bound to an amino acid residue which is on the surfaceof the protein and/or away from the surface that contacts the targetligand. In one embodiment, the combined or total molecular mass of PEGin PEG-binding polypeptide is from about 3,000 Da to 60,000 Da,optionally from about 10,000 Da to 36,000 Da. In a one embodiment, thePEG in pegylated binding polypeptide is a substantially linear,straight-chain PEG.

In one embodiment of the invention, the PEG in pegylated bindingpolypeptide is not hydrolyzed from the pegylated amino acid residueusing a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8to 16 hours at room temperature, and is thus stable. In one embodiment,greater than 80% of the composition is stable mono-PEG-bindingpolypeptide, more preferably at least 90%, and most preferably at least95%.

In another embodiment, the pegylated binding polypeptides of theinvention will preferably retain at least 25%, 50%, 60%, 70% least 80%,85%, 90%, 95% or 100% of the biological activity associated with theunmodified protein. In one embodiment, biological activity refers to itsability to bind to VEGFR-2, as assessed by KD, k_(on), or k_(off). Inone specific embodiment, the pegylated binding polypeptide protein showsan increase in binding to VEGFR relative to unpegylated bindingpolypeptide.

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

5. Therapeutic Formulations and Modes of Administration

The present invention features methods for treating conditions orpreventing pre-conditions which respond to an inhibition of VEGFbiological activity. Preferred examples are conditions that arecharacterized by inappropriate angiogenesis. Techniques and dosages foradministration vary depending on the type of specific polypeptide andthe specific condition being treated but can be readily determined bythe skilled artisan. In general, regulatory agencies require that aprotein reagent to be used as a therapeutic is formulated so as to haveacceptably low levels of pyrogens. Accordingly, therapeutic formulationswill generally be distinguished from other formulations in that they aresubstantially pyrogen free, or at least contain no more than acceptablelevels of pyrogen as determined by the appropriate regulatory agency(e.g., FDA).

Therapeutic compositions of the present invention may be administeredwith a pharmaceutically acceptable diluent, carrier, or excipient, inunit dosage form. Administration may be parenteral (e.g., intravenous,subcutaneous), oral, or topical, as non-limiting examples. In addition,any gene therapy technique, using nucleic acids encoding thepolypeptides of the invention, may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous or parenteral administration; gel, lotion,ointment, cream, or a polymer or other sustained release vehicle forlocal administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or less frequently(e.g., once every other day, once or twice weekly, or monthly). Inaddition, as is known in the art, adjustments for age as well as thebody weight, general health, sex, diet, time of administration, druginteraction, and the severity of the disease may be necessary, and willbe ascertainable with routine experimentation by those skilled in theart.

6. Exemplary Uses

The VEGFR-2 binding proteins described herein and their related variantsare useful in a number of therapeutic and diagnostic applications. Theseinclude the inhibition of the biological activity of VEGF by competingfor or blocking the binding to a VEGFR-2 as well as the delivery ofcytotoxic or imaging moieties to cells, preferably cells expressingVEGFR-2.

The small size and stable structure of these molecules can beparticularly valuable with respect to manufacturing of the drug, rapidclearance from the body for certain applications where rapid clearanceis desired or formulation into novel delivery systems that are suitableor improved using a molecule with such characteristics.

On the basis of their efficacy as inhibitors of VEGF biologicalactivity, the polypeptides of the invention are effective against anumber of conditions associated with inappropriate angiogenesis,including but not limited to autoimmune disorders (e.g., rheumatoidarthritis, inflammatory bowel disease or psoriasis); cardiac disorders(e.g., atherosclerosis or blood vessel restenosis); retinopathies (e.g.,proliferative retinopathies generally, diabetic retinopathy, age-relatedmacular degeneration or neovascular glaucoma), renal disease (e.g.,diabetic nephropathy, malignant nephrosclerosis, thromboticmicroangiopathy syndromes; transplant rejection; inflammatory renaldisease; glomerulonephritis; mesangioproliferative glomerulonephritis;haemolytic-uraemic syndrome; and hypertensive nephrosclerosis);hemangioblastoma; hemangiomas; thyroid hyperplasias; tissuetransplantations; chronic inflammation; Meigs's syndrome; pericardialeffusion; pleural effusion; autoimmune diseases; diabetes;endometriosis; chronic asthma; undesirable fibrosis (particularlyhepatic fibrosis) and cancer, as well as complications arising fromcancer, such as pleural effusion and ascites. Preferably, theVEGFR-binding polypeptides of the invention can be used for thetreatment of prevention of hyperproliferative diseases or cancer and themetastatic spread of cancers. Non-limiting examples of cancers includebladder, blood, bone, brain, breast, cartilage, colon kidney, liver,lung, lymph node, nervous tissue, ovary, pancreatic, prostate, skeletalmuscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid,trachea, urogenital tract, ureter, urethra, uterus, or vaginal cancer.Additional treatable conditions can be found in U.S. Pat. No. 6,524,583,herein incorporated by reference. Other references describing uses forVEGFR-2 binding polypeptides include: McLeod D S et al., InvestOphthalmol Vis Sci. 2002 February; 43(2):474-82; Watanabe et al. ExpDermatol. 2004 November; 13(11):671-81; Yoshiji H et al., Gut. 2003September; 52(9):1347-54; Verheul et al., Oncologist. 2000; 5 Suppl1:45-50; Boldicke et al., Stem Cells. 2001; 19(1):24-36.

As described herein, angiogenesis-associated diseases include, but arenot limited to, angiogenesis-dependent cancer, including, for example,solid tumors, blood born tumors such as leukemias, and tumor metastases;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; inflammatorydisorders such as immune and non-immune inflammation; chronic articularrheumatism and psoriasis; ocular angiogenic diseases, for example,diabetic retinopathy, retinopathy of prematurity, macular degeneration,corneal graft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation and wound healing; telangiectasia psoriasisscleroderma, pyogenic granuloma, coronary collaterals, ischemic limbangiogenesis, corneal diseases, rubeosis, arthritis, diabeticneovascularization, fractures, vasculogenesis, hematopoiesis.

A VEGFR-2 binding polypeptide can be administered alone or incombination with one or more additional therapies such as chemotherapyradiotherapy, immunotherapy, surgical intervention, or any combinationof these. Long-term therapy is equally possible as is adjuvant therapyin the context of other treatment strategies, as described above.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject therapeutic agents of the inventioncan be used alone. Alternatively, the subject agents may be used incombination with other conventional anti-cancer therapeutic approachesdirected to treatment or prevention of proliferative disorders (e.g.,tumor). For example, such methods can be used in prophylactic cancerprevention, prevention of cancer recurrence and metastases aftersurgery, and as an adjuvant of other conventional cancer therapy. Thepresent invention recognizes that the effectiveness of conventionalcancer therapies (e.g., chemotherapy, radiation therapy, phototherapy,immunotherapy, and surgery) can be enhanced through the use of a subjectpolypeptide therapeutic agent.

A wide array of conventional compounds have been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a polypeptide therapeutic agent of the present invention isadministered in combination with another conventional anti-neoplasticagent, either concomitantly or sequentially, such therapeutic agent maybe found to enhance the therapeutic effect of the anti-neoplastic agentor overcome cellular resistance to such anti-neoplastic agent. Thisallows decrease of dosage of an anti-neoplastic agent, thereby reducingthe undesirable side effects, or restores the effectiveness of ananti-neoplastic agent in resistant cells.

Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

Certain chemotherapeutic anti-tumor compounds may be categorized bytheir mechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disrupters such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (e.g., VEGF inhibitors, fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylpednisolone, prednisone, and prenisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disrupters.

In certain embodiments, pharmaceutical compounds that may be used forcombinatory anti-angiogenesis therapy include: (1) inhibitors of releaseof “angiogenic molecules,” such as bFGF (basic fibroblast growthfactor); (2) neutralizers of angiogenic molecules, such as an anti-βbFGFantibodies; and (3) inhibitors of endothelial cell response toangiogenic stimuli, including collagenase inhibitor, basement membraneturnover inhibitors, angiostatic steroids, fungal-derived angiogenesisinhibitors, platelet factor 4, thrombospondin, arthritis drugs such asD-penicillamine and gold thiomalate, vitamin D₃ analogs,alpha-interferon, and the like. For additional proposed inhibitors ofangiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89-118(1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab.Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885, 5,112,946,5,192,744, 5,202,352, and 6573256. In addition, there are a wide varietyof compounds that can be used to inhibit angiogenesis, for example,endostatin protein or derivatives, lysine binding fragments ofangiostatin, melanin or melanin-promoting compounds, plasminogenfragments (e.g., Kringles 1-3 of plasminogen), tropoin subunits,antagonists of vitronectin α_(v)β₃, peptides derived from Saposin B,antibiotics or analogs (e.g., tetracycline, or neomycin),dienogest-containing compositions, compounds comprising a MetAP-2inhibitory core coupled to a peptide, the compound EM-138, chalcone andits analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos.6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810,6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103,6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.

Depending on the nature of the combinatory therapy, administration ofthe polypeptide therapeutic agents of the invention may be continuedwhile the other therapy is being administered and/or thereafter.Administration of the polypeptide therapeutic agents may be made in asingle dose, or in multiple doses. In some instances, administration ofthe polypeptide therapeutic agents is commenced at least several daysprior to the conventional therapy, while in other instances,administration is begun either immediately before or at the time of theadministration of the conventional therapy.

The VEGFR-2 binding proteins described herein can also be detectablylabeled and used to contact cells expressing VEGFR-2 for imagingapplications or diagnostic applications. For diagnostic purposes, thepolypeptide of the invention is preferably immobilized on a solidsupport. Preferred solid supports include columns (for example, affinitycolumns, such as agarose-based affinity columns), microchips, or beads.

In one example of a diagnostic application, a biological sample, such asserum or a tissue biopsy, from a patient suspected of having a conditioncharacterized by inappropriate angiogenesis is contacted with adetectably labeled polypeptide of the invention to detect levels ofVEGFR-2. The levels of VEGFR-2 detected are then compared to levels ofVEGFR-2 detected in a normal sample also contacted with the labeledpolypeptide. An increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% in the levels of the VEGFR-2 may be considered a diagnosticindicator of a condition characterized by inappropriate angiogenesis.

In certain embodiments, the VEGFR-2 binding polypeptides of theinvention are further attached to a label that is able to be detected(e.g., the label can be a radioisotope, fluorescent compound, enzyme orenzyme co-factor). The active moiety may be a radioactive agent, suchas: radioactive heavy metals such as iron chelates, radioactive chelatesof gadolinium or manganese, positron emitters of oxygen, nitrogen, iron,carbon, or gallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁵I, ¹³¹I,¹³²I, or ⁹⁹Tc. A binding agent affixed to such a moiety may be used asan imaging agent and is administered in an amount effective fordiagnostic use in a mammal such as a human and the localization andaccumulation of the imaging agent is then detected. The localization andaccumulation of the imaging agent may be detected by radioscintigraphy,nuclear magnetic resonance imaging, computed tomography or positronemission tomography. Immunoscintigraphy using VEGFR-2 bindingpolypeptides directed at VEGFR may be used to detect and/or diagnosecancers and vasculature. For example, any of the binding polypeptideagainst the VEGFR-2 marker labeled with ⁹⁹Technetium, ¹¹¹Indium, or¹²⁵Iodine may be effectively used for such imaging. As will be evidentto the skilled artisan, the amount of radioisotope to be administered isdependent upon the radioisotope. Those having ordinary skill in the artcan readily formulate the amount of the imaging agent to be administeredbased upon the specific activity and energy of a given radionuclide usedas the active moiety. Typically 0.1-100 millicuries per dose of imagingagent, preferably 1-10 millicuries, most often 2-5 millicuries areadministered. Thus, compositions according to the present inventionuseful as imaging agents comprising a targeting moiety conjugated to aradioactive moiety comprise 0.1-100 millicuries, in some embodimentspreferably 1-10 millicuries, in some embodiments preferably 2-5millicuries, in some embodiments more preferably 1-5 millicuries.

The VEGFR-2 binding polypeptides of the present invention can also beused to deliver additional therapeutic agents (including but not limitedto drug compounds, chemotherapeutic compounds, and radiotherapeuticcompounds) to a cell or tissue expressing VEGFR-2. In one example, theVEGFR-2 binding polypeptide is fused to a chemotherapeutic agent fortargeted delivery of the chemotherapeutic agent to a tumor cell ortissue expressing VEGFR-2.

The VEGFR-2 binding polypeptides of the present invention are useful ina variety of applications, including research, diagnostic andtherapeutic applications. For instance, they can be used to isolateand/or purify receptor or portions thereof, and to study receptorstructure (e.g., conformation) and function.

In certain aspects, the various binding polypeptides of the presentinvention can be used to detect or measure the expression of VEGFR-2,for example, on endothelial cells (e.g., venous endothelial cells), oron cells transfected with a VEGFR-2 gene. Thus, they also have utilityin applications such as cell sorting and imaging (e.g., flow cytometry,and fluorescence activated cell sorting), for diagnostic or researchpurposes.

In certain embodiments, the binding polypeptides of fragments thereofcan be labeled or unlabeled for diagnostic purposes. Typically,diagnostic assays entail detecting the formation of a complex resultingfrom the binding of a binding polypeptide to VEGFR-2. The bindingpolypeptides or fragments can be directly labeled, similar toantibodies. A variety of labels can be employed, including, but notlimited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and4,098,876). When unlabeled, the binding polypeptides can be used inassays, such as agglutination assays. Unlabeled binding polypeptides canalso be used in combination with another (one or more) suitable reagentwhich can be used to detect the binding polypeptide, such as a labeledantibody reactive with the binding polypeptide or other suitable reagent(e.g., labeled protein A).

In one embodiment, the binding polypeptides of the present invention canbe utilized in enzyme immunoassays, wherein the subject polypeptides areconjugated to an enzyme. When a biological sample comprising a VEGFR-2protein is combined with the subject binding polypeptides, bindingoccurs between the binding polypeptides and the VEGFR-2 protein. In oneembodiment, a sample containing cells expressing a VEGFR protein (e.g.,endothelial cells) is combined with the subject antibodies, and bindingoccurs between the binding polypeptides and cells bearing a VEGFR-2protein recognized by the binding polypeptide. These bound cells can beseparated from unbound reagents and the presence of the bindingpolypeptide-enzyme conjugate specifically bound to the cells can bedetermined, for example, by contacting the sample with a substrate ofthe enzyme which produces a color or other detectable change when actedon by the enzyme. In another embodiment, the subject bindingpolypeptides can be unlabeled, and a second, labeled polypeptide (e.g.,an antibody) can be added which recognizes the subject bindingpolypeptide.

In certain aspects, kits for use in detecting the presence of a VEGFR-2protein in a biological sample can also be prepared. Such kits willinclude an VEGFR-2 binding polypeptide which binds to a VEGFR-2 proteinor portion of said receptor, as well as one or more ancillary reagentssuitable for detecting the presence of a complex between the bindingpolypeptide and the receptor protein or portions thereof. Thepolypeptide compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The binding polypeptides and/orantibodies, which can be labeled or unlabeled, can be included in thekits with adjunct ingredients (e.g., buffers, such as Tris, phosphateand carbonate, stabilizers, excipients, biocides and/or inert proteins,e.g., bovine serum albumin). For example, the binding polypeptidesand/or antibodies can be provided as a lyophilized mixture with theadjunct ingredients, or the adjunct ingredients can be separatelyprovided for combination by the user. Generally these adjunct materialswill be present in less than about 5% weight based on the amount ofactive binding polypeptide or antibody, and usually will be present in atotal amount of at least about 0.001% weight based on polypeptide orantibody concentration. Where a second antibody capable of binding tothe binding polypeptide is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Similarly, the present invention also relates to a method of detectingand/or quantitating expression of VEGFR-2, wherein a compositioncomprising a cell or fraction thereof (e.g., membrane fraction) iscontacted with a binding polypeptide which binds to a VEGFR-2 or portionof the receptor under conditions appropriate for binding thereto, andthe binding is monitored. Detection of the binding polypeptide,indicative of the formation of a complex between binding polypeptide andVEGFR-2 or a portion thereof, indicates the presence of the receptor.Binding of a polypeptide to the cell can be determined by standardmethods, such as those described in the working examples. The method canbe used to detect expression of VEGFR-2 on cells from an individual.Optionally, a quantitative expression of VEGFR-2 on the surface ofendothelial cells can be evaluated, for instance, by flow cytometry, andthe staining intensity can be correlated with disease susceptibility,progression or risk.

The present invention also relates to a method of detecting thesusceptibility of a mammal to certain diseases. To illustrate, themethod can be used to detect the susceptibility of a mammal to diseaseswhich progress based on the amount of VEGFR-2 present on cells and/orthe number of VEGFR-2-positive cells in a mammal. In one embodiment, theinvention relates to a method of detecting susceptibility of a mammal toa tumor. In this embodiment, a sample to be tested is contacted with abinding polypeptide which binds to a VEGFR-2 or portion thereof underconditions appropriate for binding thereto, wherein the sample comprisescells which express VEGFR-2 in normal individuals. The binding and/oramount of binding is detected, which indicates the susceptibility of theindividual to a tumor, wherein higher levels of receptor correlate withincreased susceptibility of the individual to a tumor.

EXAMPLES

The following examples are for the purposes of illustrating theinvention, and should not be construed as limiting.

Example 1 Initial Identification of KDR Binding Molecules

A library of approximately 10¹³ RNA-protein fusion variants wasconstructed based on the scaffold of the tenth type 3 domain of humanfibronectin with three randomized regions at positions 23-29, 52-55 and77-86 (amino acid nos. are referenced to SEQ ID NO:5) (three looplibrary; Xu et al, Chemistry & Biology 9:933-942, 2002). Similarlibraries were constructed containing randomized regions only atpositions 23-29 and 77-86 (two loop library) or only at positions 77-86(one loop library). A mixture of these three libraries was used for invitro selection against the extracellular domain of human VEGFR-2 (KDR,extracellular domain, residues 1-764 fused to human IgG1 Fc). For thepurposes of this application, the amino acid positions of the loops willbe defined as residues 23-30 (BC Loop), 52-56 (DE Loop) and 77-87 (FGLoop). The target binding population was analyzed by DNA sequencingafter six rounds of selection and was found to be diverse, with somereplicates present. Proteins encoded by fifteen independent clones werescreened for binding to KDR, (FIG. 1A) and the best binders weresubsequently analyzed for inhibition of target binding in the presenceof VEGF (FIG. 1B). Multiple clones were identified that inhibitedKDR-VEGF binding, suggesting that these clones bound KDR at or near thenatural ligand (VEGF) binding site. The ability of two of the bindingmolecules (VR28 and VR12) to directly inhibit VEGF-KDR interaction wasevaluated in a BIAcore assay using immobilized VEGF and a mobile phasecontaining KDR-Fc with or without a selected binding protein. VR28 and,to a lesser extent, VR12, but not a non-competing clone (VR17),inhibited KDR binding to VEGF in a dose dependent manner (FIG. 1C).Finally, in addition to binding to purified recombinant KDR, VR28 alsoappeared to bind to KDR-expressing recombinant CHO cells, but not tocontrol CHO cells (FIG. 1D).

The sequence of the binding loops of the VR28 clone is shown in thefirst row of Table 4.

While VR28 was not the most abundant clone in the sequenced bindingpopulation (one copy out of 28 sequenced clone), its binding affinity toKDR was the best among the tested clones from this binding population,with a dissociation constant of 11-13 nM determined in a radioactiveequilibrium binding assay (FIG. 3 and Table 5) and BIAcore assays (Table7). There were no changes from wild type ¹⁰Fn3 in the remaining scaffoldportion of the molecule (following correction of an incidental scaffoldchange at position 69 that had no effect on binding). However, VR28showed little inhibition of VEGF-KDR signaling in a VEGF-dependent cellproliferation assay. Thus, while the selection from the naïve libraryyielded antibody mimics that interfered with the interaction betweenVEGF and KDR in biochemical binding studies, affinity improvements wereuseful for neutralizing function in a biological signal transductionassay.

Example 2 Affinity Maturation of Clone VR28

A mutagenesis strategy focusing on altering sequences only in thebinding loops was employed. To initially test which loops were morelikely to result in improvement, loop-directed hypermutagenic PCR wascarried out to introduce up to 30% mutations independently into eachloop of VR28. After three rounds of selection against KDR, multipleclones with improved binding to KDR-Fc were observed. Sequence analysisof the selection pools revealed that the majority of mutations wereaccumulated in the FG loop while the BC and DE loops remained almostintact. This result indicated that the FG loop was the most suitabletarget for further modification.

Consequently, a new library of approximately 10¹² variants wasconstructed by altering the sequence of VR28 in the FG loop usingoligonucleotide mutagenesis. For each of the FG loop positions (residues77-86 [VAQNDHELIT (SEQ ID NO:198)] as well as the following Proline[residue 87]), a 50:50 mixture of the VR28-encoding DNA and NNS wasintroduced at each position. DNA sequence analysis of a random sample ofapproximately 80 clones revealed an average of six amino acid changesper clone as expected. Lower KDR-Fc concentrations were utilized duringselection to favor clones with better affinities to the target. Theprofile of target binding during the four rounds of selection is shownin FIG. 2. After four rounds of selection the binding population wassubcloned and analyzed. Table 5 and FIG. 3A summarize affinitymeasurements of individual binding clones. The measured bindingconstants to KDR-Fc ranged from <0.4 to <1.8 nM, a 10-30 improvementover VR28 (11 nM).

Sequence analysis, some of which is shown in Table 4 (K clones),revealed that while the binding population was diverse, severalconsensus motifs could be identified among the clones. Most noticeably,Pro87 and Leu84 were found in nearly all clones (as in VR28), suggestingthat these residues may be essential for the structure of the bindingsite. A positively charged amino acid at position 82 appears to berequired since only H82K or H82R changes were seen in the sequencedclones and an aliphatic amino acid was predominant at position 78. D81was often mutated to a G, resulting in the loss of negative charge atthis position and a gain in flexibility. In addition, the overallmutation rate in the selected population was comparable to the poolprior to selection, which suggested that the FG loop is very open tochanges.

Several residues in the N-terminus of the ¹⁰Fn3 domain of humanfibronectin are located in close proximity to the FG loop, as suggestedby structural determinations (Main et al, Cell 71:671-678, 1992). Theclose proximity of the two regions could potentially have a negativeimpact on target binding. Two incidental mutations in the N-terminalregion, L8P and L8Q, resulted in better binding to KDR in a number ofselected clones, presumably due to a change of the location of theN-terminus relative to the FG loop. To further test the impact of theN-terminus, we created binding molecules for 23 different KDR binders inwhich the N-terminal first eight residues before the β-sheet weredeleted. We then compared target binding to the non-deletedcounterparts. On average, binding to KDR-Fc was about 3-fold better withthe deletion, as shown in FIG. 3B.

Example 3 Selection of Binders with Dual Specificities to Human (KDR)and Mouse (Flk-1) VEGFR-2

VR28 and most of the affinity matured variants (K clones) failed to bindthe mouse homolog of KDR, Flk1, as shown in FIG. 4. However, since KDRand Flk1 share a high level of sequence identity (85%, Claffey et al.,J. Biol. Chem. 267:16317-16322 (1992), Shima et al., J. Biol. Chem.271:3877-3883 (1996)), it is conceivable to isolate antibody mimics thatcan bind both KDR and Flk1. Such dual binders were desirable becausethey would allow the same molecule to be tested in functional studies inanimal models and subsequently in humans.

The population of clones following FG loop mutagenesis and selectionagainst KDR for four rounds was further selected against Flk1 for anadditional three rounds. As shown in FIG. 2 an increase in binding toFlk1 was observed from Round 5 to Round 7, indicating enrichment of Flk1binders. Analysis of binding for multiple individual clones revealedthat in contrast to the clones selected against KDR only (K clones),most clones derived from additional selection against Flk1 (E clones)are able to interact with both KDR and Flk1. The binding constants toboth targets, as determined using a radioactive equilibrium bindingassay (Table 6 and FIG. 5) and BIAcore (Table 7), indicate thatindividual clones were able to bind both targets with high affinities.

For example, E19 has a Kd of 60 pM to KDR, and 340 pM to Flk-1. Theseresults demonstrate that a simple target switch strategy in theselection process, presumably through selection pressures exerted byboth targets, has allowed the isolation of molecules with dual bindingspecificities to both KDR and Flk-1 from a mutagenized population ofVR28, a moderate KDR binder that was not able to bind Flk-1. Theselected fibronectin-based binding proteins are highly specific toVEGFR-2 (KDR) as no substantial binding to VEGFR1 was observed at hightarget concentration.

Sequence analysis revealed some motifs similar to those observed in theKDR binder pool (Leu and Pro at residues 84 and 87 respectively;positively charged amino acid at residue 82, predominantly Arg) and somethat were not maintained (aliphatic at position 78). In addition, themotif ERNGR (residues 78-82) was present in almost all clones binding toFlk-1 (Table 4); this motif was barely discernable in the KDR bindingpool. R79 and R82 appear to be particularly important for high affinitybinding to Flk-1, since binding to Flk-1, but not KDR, is greatlyreduced when a different residue is present at this position (FIG. 6A).To determine the importance of each loop in binding to KDR and Flk-1,the loops of clones E6 and E26 shown in Table 4, were substituted oneloop at a time by NNS randomized sequence. As shown in FIG. 6B, afterthe substitution, the proteins are no longer able to bind either KDR orFlk-1. These results indicate that each loop is required for binding tothe targets, suggesting a cooperative participation of all three loopsin interacting with the targets.

An alternative mutagenesis strategy was independently employed toproduce clones capable of binding to both targets. The clone 159Q(8)L(Table 4), the product of hypermutagenic PCR affinity maturation of VR28that binds KDR with high affinity (Kd=2 nM; Table 7) and Flk-1 with pooraffinity (Kd□3000 nM), was chosen as a starting point. The first sixamino acids of the FG loop were fully randomized (NNS), leaving thefollowing five residues (ELFTP) intact. After six rounds of selectionagainst Flk-1, the binding pool was re-randomized at the DE loop(positions 52-56) and the selection was performed for three additionalrounds against Flk-1 and one round against KDR. A number of highaffinity binding molecules to both KDR and Flk-1 were thus obtained(Tables 4 and FIG. 4). For example, clone M5FL, while retaining highbinding affinity to KDR (Kd=890 pM), can bind Flk-1 at a Kd of 2.1 nM, a1000-fold improvement over the original clone. Interestingly, the ERNGRmotif, found in Flk-1 binding molecules selected from a mutagenizedpopulation of VR28, was also present in multiple clones derived fromclone 159Q(8)L mutagenesis and selection, despite a full randomizationof this region of the FG loop. The isolation of similar bindingmolecules from two independent libraries suggests that the affinitymaturation process is robust for isolating optimal Flk-1 binding motifslocated in the FG loop.

Example 4 Cell Surface Binding and Neutralization of VEGF Activity InVitro

The functionality of KDR and Flk-1 binding molecules in a cell culturemodel system was evaluated with E. coli produced binding molecules.Using a detection system consisting of anti-His6 tag murine antibody(the E. coli expressed proteins were expressed with a His tag) and ananti-murine fluorescently labeled antibody the binding molecules wereshown to bind specifically to mammalian cells expressing KDR or Flk-1with low nanomolar EC50s, (FIG. 7 and Table 8).

More importantly, using recombinant BA/F3 cells (DSMZ-Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH) expressing the extracellularKDR or Flk-1 domain linked to the erythropoietin receptor signalingdomain, these molecules inhibited VEGF-stimulated cell proliferation ina dose dependent fashion, with IC50 3-12 mM for KDR expressing cells,and 2-5 nM for Flk-1 expressing cells. The potency of inhibition appearsto be similar to control anti-KDR and anti-Flk-1 monoclonal antibodies,as shown in FIG. 8 and Table 9.

A number of clones were further tested for VEGF-inhibition of the growthof HUVEC cells (Human Umbilical Vein Endothelial Cells). HUVEC cells arenatural human cells that are closely related to cells in the body thatrespond to VEGF. As shown in FIG. 9 and Table 10, the fibronectin-basedbinding proteins were also active in inhibiting VEGF activity in thishuman-derived cell system while the wild type fibronectin-based scaffoldprotein was inactive.

Example 5 Thermal Stability and Reversible Refolding of M5FL Protein

The thermal stability of KDR-binder M5FL was established usingdifferential scanning calorimetry (DSC). Under standard PBS bufferconditions (sodium phosphate pH 7.4, 150 mM NaCl), M5FL was found tohave a single non-reversible thermal melting transition at 56° C.Subsequently, sodium acetate pH 4.5 was identified as a favorable bufferfor M5FL protein solubility. DSC experiments in this buffer (100 mM)demonstrated that M5FL is more stable under these conditions (Tm=67-77°C.) and that the melting transition is reversible (FIG. 10). Reversiblethermal transitions have been used to identify favorable conditions thatsupport long-term storage of protein therapeutics (Remmele et al,Biochemistry 38:5241 (1999), so Na-acetate pH 4.5 has been identified asan optimized buffer for storing the M5FL protein.

Example 6 In Vitro Binding and Cell-Based Activity of PEGylated M5FLProtein

The M5FL protein was produced in an E. coli expression system with aC-terminal extension to yield the following protein sequence (C-terminalextension underlined with Cys100 shaded; a significant percentage ofprotein is produced with the initial methionine removed): (SEQ IDNO:199) MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPLATISGLKPGVDYTITVYAVTKERNGRELFTPISINYRTEIDK PCQHHHHHH

The single sulfhydryl of the cysteine residue at position 100 was usedto couple to PEG variants using standard maleimide chemistry to yieldtwo different PEGylated forms of M5FL. Both a linear 20 kD PEG and abranched 40 kD PEG (Shearwater Corporation) were conjugated to M5FL toproduce M5FL-PEG20 and M5FL-PEG40, respectively. The PEGylated proteinforms were purified from unreacted protein and PEG by cation exchangechromatography. Covalent linkage of the two PEG forms of M5FL wasverified by SDS-PAGE (FIG. 11) and mass spectroscopy.

In vitro affinity measurements were made using surface plasmon resonance(SPR) (BIAcore) with both the human and mouse VEGF-receptor targetproteins immobilized via amide chemistry on the BIAcore chip. For bothtarget proteins, both the 20 and 40 kD PEGylated M5FL forms were foundto have slower on-rates (ka) relative to unmodified M5FL with littleeffect on off-rates (kd; Table 11).

The functionality of the PEGylated M5FL preparations was tested usingthe Ba/F3 system described in Example 4. FIG. 12 shows a plot of A490(representing the extent of cell proliferation) as a function ofconcentration of each of the binders. The curves were nearly identical,indicating there was little effect of PEGylation on the biologicalactivity of either of the PEGylated forms.

The k_(on), k_(off) and K_(D) were analyzed for a subset of KDR-bindingpolypeptides and compared to the EC50 for the BaF3 cell-based VEGFinhibition assay. Scatter plots showed that the kon was well-correlatedwith the EC50, while koff was poorly correlated. Greater than 90% ofKDR-binding proteins with a kon of 105s-1 or greater had an EC50 of 10nM or less. KD is a ratio of kon and koff, and, as expected, exhibits anintermediate degree of correlation with EC50.

Many of the KDR-binding proteins, including CT-01, were assessed forbinding to VEGFR-1, VEGFR-2 and VEGFR-3. The proteins showed a highdegree of selectivity for VEGFR-2.

Example 6 Preparation of KDR Binding Protein CT-01 Blocks VEGFR-2Signaling in Human Endothelial Cells

Following the methodologies described in the preceding Examples,additional ¹⁰Fn3-based KDR binding proteins were generated. As describedfor the development of the M5FL protein in Example 5, above, proteinswere tested for K_(D) against human KDR and mouse Flk-1 using theBIAcore binding assay and for IC50 in a Ba/F3 assay. A protein termedCT-01 exhibited desirable properties in each of these assays and wasused in further analysis.

The initial clone from which CT-01 was derived had a sequence:GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGWNGRLLSIPISINYRT (SEQ ID NO:200). The FG loop sequence isunderlined.

Affinity maturation as described above produced a core form of CT-01:(SEQ ID NO:192) EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT.

The CT-01 molecule above has a deletion of the first 8 amino acids andmay include additional amino acids at the N- or C-termini. For example,an additional MG sequence may be placed at the N-terminus. The M willusually be cleaved off, leaving a GEV . . . sequence at the N-terminus.The re-addition of the normal 8 amino acids at the N-terminus alsoproduces a KDR binding protein with desirable properties. The N-terminalmethionine is generally cleaved off to yield a sequence: (SEQ ID NO:193)VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT.

For use in vivo, a form suitable for PEGylation may be generated. Forexample, a C-terminal tail comprising a cysteine was added andexpressed, as shown below for a form lacking the eight N-terminal aminoacids. GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPCQ (SEQ ID NO:194). The PEGylatedform of this molecule is used in the in vivo experiments describedbelow. A control form with a serine instead of a cysteine was also used:(SEQ ID NO:195) GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPSQ.

The same C-terminal tails may also be added to CT-01 forms having theN-terminal eight amino acids, such as is shown in SEQ ID NO:193.

Additional variants with desirable KDR binding properties were isolated.The following core sequence has a somewhat different FG loop, and may beexpressed with, for example, an N-terminal MG sequence, an N-terminalsequence that restores the 8 deleted amino acids, and/or a C-terminaltail to provide a cysteine for PEGylation.EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT (SEQ ID NO:196). Another such variant hasthe core sequence: (SEQ ID NO:197)VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT.

A comparison of these variants shows a consensus sequence for the FGloop of: D/E)GXNXRXXIP (SEQ ID NO:3). With greater particularity, theconsensus sequence may be expressed as (D/E)G(R/P)N(G/E)R(S/L)(S/F)IP(SEQ ID NO:4).

Example 7 CT-01 Blocks VEGFR-2 Signaling in Human Endothelial Cells

As shown in FIG. 13, VEGF-A signaling through VEGFR-2 is mediated byphosphorylation of the intracellular domain of VEGFR-2, followed byactivation of pathway involving phospholipase C gamma (PLCγ), ProteinKinase C (PKC), Raf-1, MEK1/2, ERK1/2, leading to endothelial cellproliferation.

To assess whether KDR binders disclosed herein inhibited activation ofthis signaling pathway, Human Microvascular Endothelial Cells weretreated with a VEGFR binding polypeptide (e.g., CT-01) for 30 min andstimulated with VEGF-A for 5 min. Total cell lysates were analyzed bySDS-PAGE and western analysis, using antibodies specific tophospho-VEGFR-2, non-phospho-VEGFR-2, phosphor-ERK1/2 andnon-phospho-ERK1/2.

As shown in FIG. 13, 130 pM CT-01 inhibits formation of phosphor-VEGFR-2and also decreases the formation of the downstream phosphorylatedERK1/2. Phosphorylated ERK1/2 is not entirely eliminated, probably dueto the fact that ERK1/2 receives signals from a number of additionalsignaling pathways.

Example 8 Fibronectin-based KDR Binding Proteins Disrupt Signaling byVEGF-A and VEGF-D

VEGFR-2 is a receptor for three VEGF species, VEGF-A, VEGF-C and VEGF-D.Experiments were conducted to evaluate the effects of fibronectin-basedKDR binding proteins on VEGF-A and VEGF-D mediated signaling throughKDR.

A Ba/F3 cell line dependent on Flk-1 mediated signaling was generated.As shown in the left panel of FIG. 14, cell viability could bemaintained by treating the cells with VEGF-A or VEGF-D, althoughsignificantly higher levels of VEGF-D were required.

As shown in the middle panel of FIG. 14, cells were maintained in thepresence of 15 ng/ml of VEGF-A and contacted with the M5FL or CT-01proteins disclosed herein, or with the DC-101 anti-Flk-1 antibody. Eachreagent reversed the VEGF-A-mediated cell viability, indicating thatVEGF-A signaling through Flk-1 was blocked.

As shown in the right panel of FIG. 14, cells were maintained in thepresence of 300 ng/ml of VEGF-D and contacted with the M5FL or F10proteins disclosed herein, or with an anti-VEGF-A antibody. M5FL and F10reversed the VEGF-D-mediated cell viability, indicating that VEGF-Dsignaling through Flk-1 was blocked. The anti-VEGF-A antibody had noeffect, demonstrating the specificity of the assay.

Example 9 Pharmacokinetics

Pharmacokinetic Studies: Native CT-01 or a pegylated form (40 kDa PEG,CT-01PEG40) were iodinated with ¹²⁵I. 10-20 mCi of iodinated proteinswere injected into adult male rats either i.v. or i.p. and iodinatedproteins levels were determined at the indicated times. For tissuedistribution studies, rats were sacrificed at 15 min, 2 hr and 6 hr andradioactivity levels determined. See FIGS. 15 and 16. Unmodified CT-01is a 12 kDa protein that is rapidly cleared from the blood. Thearea-under-curve value (AUC) value is 14.6 hr*mg/mL with a clearance of69.9 mL/hr/kg, a maximum serum concentration of 9.1 mg/ml. The initialhalf-life (α) is 0.3 hours and the second phase half-life (β) is 13.5hours. By comparison, i.v. PEGylated CT-01 has greatly increasedpresence in the blood, mostly because of a dramatic decrease in theinitial phase of clearance. The AUC is increased greater than 10 fold to193, the clearance rate is decreased by greater than 10 fold to 5.2, theCmax is 12.9 mg/mL. The α half-life is increased to 1 hour, and the β isincreased to 16.2 hours. These pharmacokinetics in rats are equivalentto a twice-weekly dosing regimen in humans, a rate of dosing that iswell within acceptable ranges.

Intraperitoneal (i.p.) administration of PEGylated CT-01 hadreservoir-like pharmacokinetics. There was no initial spike in the bloodconcentration of CT-01. Instead, the amount of CT-01 built up moreslowly and decreased slowly. Such pharmacokinetics may be desirablewhere there is concern about side effects from the initial spike inCT-01 concentration upon intravenous administration. It is likely thatother ¹⁰FN3-based agents would exhibit similar behavior in i.p.administration. Accordingly, this may be a generalizable mode forachieving a time-delayed dosing effect with ¹⁰Fn3-based agents.

As shown in FIG. 16, the liver is the primary route for secretion of thePEGylated form of CT-01. No long term accumulation of CT-01 wasdetected.

Similar results were obtained using a CT-01 conjugated to a 20 kDa PEGmoiety.

Example 10 In Vivo Efficacy of CT-01

The Miles assay, as outlined in FIG. 17, is used to evaluate Dose,Schedule and Administration parameters for the tumor efficacy studies.Balb/c female mice were injected i.p. with buffer or CT-01PEG40 at 1, 5and 20 mg/kg 4 hr prior to VEGF challenge. Intradermal focaladministration of VEGF-A into the back skin induces vessel leakage ofEvans blue dye (FIGS. 17 and 18).

Mice treated with a KDR binding agent showed a statistically significantdecrease in the level of VEGF-mediated vessel leakage. Both 5 mg/kg and20 mg/kg dosages with CT-01 showed significant results. Therefore, a 5mg/kg dosage was selected for mouse tumor model studies.

Example 11 CT-01 Inhibits Tumor Growth

B16-F10 Murine Melanoma Tumor Assay:

2×10⁶ B16-F10 murine melanoma tumor cells were implanted subcutaneouslyinto C57/BL male mice at Day 1. At day 6 a palpable mass was detected.On day 8 when tumors were of measurable size, daily i.p. injections ofeither Vehicle control, 5, 15, or 40 mg/kg CT-01PEG40 were started. Thelowest dose 5 mg/kg decreased tumor growth. At day 18, mice treated with15 and 40 mg/kg showed 50% and 66% reduction in tumor growth. See FIG.19.

U87 Human Glioblastoma Assay:

5×10⁶ U87 human glioblastoma tumor cells were implanted subcutaneouslyinto nude male mice. When tumor volume reached approximately 50 mm3treatment started (day 0). Vehicle control, 3, 10, or 30 mg/kgCT-01PEG40 were injected i.v. every other day (EOD). The anti-Flk-1antibody DC101 was injected at 40 mg/kg twice a week as published forits optimal dose schedule. The lowest dose 3 mg/kg decreased tumorgrowth. At day 12, mice treated with 10 and 30 mg/kg showed 50%reduction in tumor growth. See FIG. 20. Effectiveness is comparable tothat of the anti-Flk-1 antibody.

The following materials and methods were used for the experimentsdescribed in Examples 1-11.

Recombinant Proteins:

Recombinant human VEGF₁₆₅, murine VEGF₁₆₄, human neurotrophin-4 (NT4),human and mouse vascular endothelial growth factor receptor-2 Fcchimeras (KDR-Fc and Flk-1-Fc) were purchased from R&D systems(Minneapolis, Minn.). Biotinylation of the target proteins was carriedout in 1×PBS at 4° C. for 2 hours in the presence of EZ-Link™Sulfo-NHS-LC-LC-Biotin (Pierce, Ill.). Excess of EZ-Link™Sulfo-NHS-LC-LC-Biotin was removed by dialysis against 1×PBS. The levelof biotinylation was determined by mass spectroscopy and target proteinconcentrations were determined using Coomassie Protein Plus Assay(Pierce, Ill.).

Primers:

The following oligonucleotides were prepared by chemical synthesis foreventual use in library construction and mutagenesis of selected clones.T7 TMV Fn: (SEQ ID NO:201) 5′ GCG TAA TAC GAC TCA CTA TAG GGA CAA TTACTA TTT ACA ATT ACA ATG GTT TCT GAT GTT CCG AGG 3′ T7 TMV N-terminusdeletion: (SEQ ID NO:202) 5′ GCG TAA TAC GAC TCA CTA TAG GGA CAA TTA CTATTT ACA ATT ACA ATG GAA GTT GTT GCT GCG ACC CCC ACC AGC CTA 3′ MK165-4A20: (SEQ ID NO:203) 5′ TTT TTT TTT TTT TTT TTT TTA AAT AGC GGA TGC CTTGTC GTC GTC GTC CTT GTA GTC 3′ N-terminus forward: (SEQ ID NO:204)5′ ATG GTT TCT GAT GTT CCG AGG GAC CTG GAA GTT GTT GCT GCG ACC CCC ACCAGC CTA CTG ATC AGC TGG 3′ BCDE reverse: (SEQ ID NO:205) 5′ AGG CAC AGTGAA CTC CTG GAC AGG GCT ATT TCC TCC TGT TTC TCC GTA AGT GAT CCT GTA ATATCT 3′ BCDE forward: (SEQ ID NO:206) 5′ AGA TAT TAC AGG ATC ACT TAC GGAGAA ACA GGA GGA AAT AGC CCT GTC CAG GAG TTC ACT GTG CCT 3′ DEFG reverse:(SEQ ID NO:207) 5′ AGT GAC AGC ATA CAC AGT GAT GGT ATA ATC AAC TCC AGGTTT AAG GCC GCT GAT GGT AGC TGT 3′ DEFG forward: (SEQ ID NO:208) 5′ ACAGCT ACC ATC AGC GGC CTT AAA CCT GGA GTT GAT TAT ACC ATC ACT GTG TAT GCTGTC ACT 3′ C-terminus polyA: (SEQ ID NO:209) 5′ TTT TTT TTT TTT TTT TTTTAA ATA GCG GAT GCC TTG TCG TCG TCG TCC TTG TAG TCT GTT CGG TAA TTA ATGGAA AT 3′ Hu3′FLAGSTOP: (SEQ ID NO:210) 5′ TTT TAA ATA GCG GAT GCC TTGTCG TCG TCG TCC TTG TAG TCT GTT CGG TAA TTA ATG G 3′ VR28FG-50: (SEQ IDNO:211) 5′ GTG TAT GCT GTC ACT 123 145 463 665 165 465 163 425 625 645447 ATT TCC ATT AAT TAC 3′,, where 1 = 62.5% G + 12.5% A + 12.5% T+ 12.5% C; 2 = 2.5% G + 12.5% A + 62.5% T + 12.5% C; 3 = 75% G + 25% C;4 = 12.5% G + 12.5% A + 12.5% T 62.5% C; 5 = 25% G + 75% C; 6 = 12.5% G+ 62.5% A + 12.5% T + 12.5% C; 7: 25% G + 50% A + 25% C F1U2: (SEQ IDNO:212) 5′ TAA TAC GAC TCA CTA TAG GGA CAA TTA CTA TTT ACA ATT CTA TCAATA CAA TGG TGT CTG ATG TG CCG 3′ F2: (SEQ ID NO:213) 5′ CCA GGA GAT CAGCAG GGA GGT CGG GGT GGC AGC CAC CAC TTC CAG GTC GCG CGG CAC ATC AGA CACCAT TGT 3′ F3159: (SEQ ID NO:214) 5′ ACC TCC CTG CTG ATC TCC TGG CGC CATCCG CAT TTT CCG ACC CGC TAT TAC CGC ATC ACT TAC G 3′ F4: (SEQ ID NO:215)5′ CAC AGT GAA CTC CTG GAC CGG GCT ATT GCC TCC TGT TTC GCC GTA AGT GATGCG GTA ATA GCG 3′ F5159: (SEQ ID NO:216) 5′ CGG TCC AGG AGT TCA CTG TGCCGC TGC AGC CGC CGG CGG CTA CCA TCA GCG GCC TTA AAC C 3′ F5-X5: (SEQ IDNO:217) 5′ CG GTC CAG GAG TTC ACT GTG CCG NNS NNS NNS NNS NNS GCT ACCATC AGC GGC CTT AAA CC 3′ F6: (SEQ ID NO:218) 5′ AGT GAC AGC ATA CAC AGTGAT GGT ATA ATC AAC GCC AGG TTT AAG GCC GCT GAT GGT AG 3′ F7X6159: (SEQID NO:219) 5′ ACC ATC ACT GTG TAT GCT GTC ACT NNS NNS NNS NNS NNS NNSGAA CTG TTT ACC CCA ATT TCC ATC AAC TAC CGC ACA GAC TAC AAG 3′ F8: (SEQID NO:220) 5′ AAA TAG CGG ATG CGC GTT TGT TCT GAT CTT CCT TAT TTA TGTGAT GAT GGT GGT GAT GCT TGT CGT CGT CGT CCT TGT AGT CTG TGC GGT AGT TGAT 3′ C2asaiA20: (SEQ ID NO:221) 5′ TTT TTT TTT TTT TTT TTT TTA AAT AGCGGA TGC GCG TTT GTT CTG ATC TTC 3′ C2RT: (SEQ ID NO:222) 5′ GCG CGT TTGTTC TGA TCT TCC 3′ hf01 BC reverse: (SEQ ID NO:223) 5′ TGCC TCC TGT TTCGCC GTA AGT GAT GCG GTA ATA GCG SNN SNN SNN SNN SNN SNN SNN CCA GCT GATCAG CAG 3′ hf01 DE reverse: (SEQ ID NO:224) 5′ GAT GGT AGC TGT SNN SNNSNN SNN AGG CAC AGT GAA CTC CTG GAC AGG GCT ATT GCC TCC TGT TTC GCC 3′hf01 FG reverse: (SEQ ID NO:225) 5′ GT GCG GTA ATT AAT GGA AAT TGG SNNSNN SNN SNN SNN SNN SNN SNN SNN SNN AGT GAC AGC ATA CAC 3′ BCDE rev:(SEQ ID NO:226) 5′ CCT CCT GTT TCT CCG TAA GTG 3′ BCDEfor: (SEQ IDNO:227) 5′ CAC TTA CGG AGA AAC AGG AGG 3′ hf01 DE-FG forward: (SEQ IDNO:228) 5′ ACA GCT ACC ATC AGC GGC CTT AAA CCT GGC GTT GAT TAT ACC ATCACT GTG TAT GCT GTC ACT 3′ Front FG reverse: (SEQ ID NO:229) 5′ AGT GACAGC ATA CAC AGT 3′ hf01 RT Flag PolyA reverse: (SEQ ID NO:230) 5′ TTTTTT TTT TTT TTT TTT TTA AAT AGC GGA TGC CTT GTC GTC GTC GTC CTT GTA GTCTGT GCG GTA ATT AAT GGA 3′ 5-RI-hKDR-1B: (SEQ ID NO:231) 5′ TAG AGA ATTCAT GGA GAG CAA GGT GCTG 3′ 3-EPO/hKDR-2312B: (SEQ ID NO:232) 5′ AGG GAGAGC GTC AGG ATG AGT TCC AAG TTC GTC TTT TCC 3′ 5-RI-mKDR-1: (SEQ IDNO:233) 5′ TAG AGA ATT CAT GGA GAG CAA GGC GCT G 3′ 3-EPO/mKDR-2312:(SEQ ID NO:234) 5′ AGG GAG AGC GTC AGG ATG AGT TCC AAG TTG GTC TTT TCC3′ 5-RI-hTrkB-1: (SEQ ID NO:235) 5′ TAG AGA ATT CAT GAT GTC GTC CTG GATAAG GT 3′ 3-EpoR/hTrkB-1310: (SEQ ID NO:236) 5′ AGG GAG AGC GTC AGG ATGAGA TGT TCC CGA CCG GTT TTA 3′ 5-hKDR/EPO-2274B: (SEQ ID NO:237) 5′ GGAAAA GAC GAA CTT GGA ACT CAT CCT GAC GCT CTC CCT 3′ 5-mKDR/EPO-2274: (SEQID NO:238) 5′ GGA AAA GAC CAA CTT GGA ACT CAT CCT GAC GCT CTC CCT 3′3-XHO-EpoR-3066: (SEQ ID NO:239) 5′ TAG ACT CGA GTC AAG AGC AAG CCA CATAGCT 3′ 5′hTrkB/EpoR-1274: (SEQ ID NO:240) 5′ TAA AAC CGG TCG GGA ACATCT CAT CCT GAC GCT CTC CCT 3′Buffers

The following buffers were utilized in the experiments described herein.Buffer A (100 mM Tris HCl, 1M NaCl, 0.05% Tween-20, pH 8.0); Buffer B(1×PBS, 0.02% Triton X100); Buffer C (100 mM Tris HCl, 60 mM EDTA, 1MNaCl, 0.05% Triton X100, pH 8.0); Buffer Ca (100 mM Tris HCl, 1M NaCl,0.05% Triton X100, pH 8.0); Buffer D (2M NaCl, 0.05% Triton); Buffer E(1×PBS, 0.05% Triton X100, pH 7.4); Buffer F (1×PBS, 0.05% Triton X100,100 mM imidazole, pH 7.4); Buffer G (50 mM HEPES, 150 mM NaCl, 0.02%Triton X-100, 1 mg/ml bovine serum albumin, 0.1 mg/ml salmon sperm DNA,pH 7.4); Buffer H (50 mM HEPES, 150 mM NaCl, 0.02% Triton X-100, pH7.4); Buffer I (1×PBS, 0.02% Triton X-100, 1 mg/ml bovine serum albumin,0.1 mg/ml salmon sperm DNA, pH 7.4); Buffer J (1×PBS, 0.02% TritonX-100, pH 7.4); Buffer K (50 mM NaH₂PO₄, 0.5 M NaCl, 5% glycerol, 5 mMCHAPS, 25 mM imidazole, 1× Complete™ Protease Inhibitor Cocktail(Roche), pH 8.0); Buffer L (50 mM NaH₂PO₄, 0.5 M NaCl, 5% glycerol, 25mM imidazole, pH 8.0); Buffer M (1×PBS, pH 7.4, 25 mM imidazole, pH7.4); Buffer N (1×PBS, 250 mM imidazole, pH 7.4); Buffer 0 (10 mM HEPES,150 mM NaCl, 0.005% Tween 20, pH 7.4).

Primary Library Construction:

The construction of the library using the tenth domain of humanfibronectin as a scaffold was previously described (Xu et al, 2002,supra). Three loop regions, corresponding to positions 23-29, 52-55, and77-86, respectively, were randomized using NNS (standard nucleotidemixtures, where N=equimolar mixture of A, G, T, C; S=equimolar mixtureof G and C) as the coding scheme. Similar libraries were constructedcontaining randomized regions only at positions 23-29 and 77-86 (twoloop library) or only at positions 77-86 (one loop library). Theselibraries were mixed in approximately equimolar amounts. This mixedlibrary contained ˜1×10¹³ clones and was used in the KDR selection thatidentified VR28.

Mutagenic Library Construction:

Hypermutagenic PCR. Scaffold mutation T(69)I in VR28 clone was correctedback to wild type sequence by PCR (see below) and no change in bindingcharacteristics of VR28 binder to KDR was observed. Mutations wereintroduced into the loop regions of VR28 using conditions describedpreviously (Vartanian et al, Nuc. Acid Res. 24:2627-2631, 1996). Threerounds of hypermutagenic PCR were conducted on a VR28 template usingprimer pairs flanking each loop (N-terminus forward/BCDE reverse, BCDEforward/DEFG reverse, DEFG forward/C-terminus polyA). The resultingfragments were assembled using overlap extension and PCR with flankingprimers T7TMV Fn and MK165-4 A20. DNA sequencing of the clones from thefinal PCR reaction confirmed correct assembly of the library. Up to 30%mutagenesis rate was observed in the loop regions, as compared to 1.5%in the scaffold regions.

Oligo mutagenesis. Oligo mutagenesis of the FG loop of VR28 by PCRutilized the VR28FG-50 primer, DEFG reverse primer and flanking primers.At each nucleotide position encoding the FG loop, primer VR28FG-50contained 50% of the VR28 nucleotide and 50% of an equimolar mixture ofall four nucleotides (N) or of G or C(S). This scheme was designed toresult in approximately 67% of the amino acids of the VR28 FG loop beingrandomly replaced by another amino acid which was confirmed by DNAsequencing.

159 (Q8L) randomized sub-libraries. Oligo mutagenesis of the FG loop ofClone 159 (Q8L) clone, a three-step extension and amplification wasperformed. For the first extension, pairs of primers (a: F1U2/F2, b:F3159/F4, c: F5159/F6, d: F7X6159/F8) were mixed in equal concentrations(100 pmol each) and amplified for 10 cycles. For the second extension,1/20 of the first reactions were combined (a/b and c/d) andamplification was continued for another 10 cycles. To bias theamplification in favor of extension rather than re-annealing of fullycomplementary fragments, a linear amplification of the half-constructproducts (0.5 pmol each) was performed for an additional 20 cycles using50 pmol of either F1U2 forward primer for fragment ab, or the C2asaiA20reverse primer for fragment cd. Finally, the extended half-constructfragments ab and cd were combined and amplified for 20 cycles withoutany additional components. Primer F7X6159 contained NNS at each of thefirst 6 coding positions of Clone 159 (Q8L) and was otherwise identicalto Clone 159 (Q8L). Correct assembly of the library 159 (Q8L)-FGX6 wasconfirmed by DNA sequencing of clones from the final PCR reaction. Thesub-library contained ˜1×10⁹ clones.

For randomization of the DE loop of post round 6 (PR6) selection pool ofthe 159 (Q8L)-FGX6 library, two half-construct fragments were preparedby PCR using primers F1U2/F4 and F5×5/C2asaiA20. The F5×5 primercontained NNS at the four positions of the DE loop as well as atposition 56. Then, the extended fragments ab and cd were combined andamplified for 20 cycles without any additional components.

Introduction of Point Mutations, Deletion and Random (NNS) LoopSequences into Fibronectin-Based Scaffold Proteins:

Scaffold mutation T(69)I of VR28 binder was corrected back to wild typesequence in two-step PCR using VR28 clone as a template. Half-constructfragments, obtained with primers N-terminus forward/DEFG reverse andDEFG forward/C-terminus polyA, were combined and the whole VR28 (169T)clone (designated as VR28 in the text) was constructed using primersT7TMV Fn and MK165-4 A20. Correction of N-terminus mutations in clone159 (Q8 to L) was performed by PCR with primers N-terminusforward/C-terminus polyA followed by extension with primers T7TMV Fn andMK165-4 A20.

Introduction of deletion Δ 1-8 into the N-terminus of fibronectin-basedscaffold proteins was performed by amplification using primers T7 TMVN-terminus deletion and MK165-4 A20.

Construction of the chimeras of E clones containing NNS loop sequenceswas performed by two-step PCR. Loop regions were amplified using primersT7 TMV N-terminus deletion/BCDE rev (a: BC loop of E clones); N-terminusforward/hf01 BC reverse (b: BC NNS); BCDE for/Front FG reverse (c: DEloop of E clones); BCDE for/hf01 DE reverse (d: DE NNS); hf01 DE-FGforward/hf01 RT-Flag PolyA reverse (e: FG of E clones); hf01 DE-FGforward/hf01 FG reverse (f: FG NNS). Fragments b/c/e, a/d/e, a/c/f werecombined and the whole pools were constructed by extension andamplification using primers T7Tmv N-terminus deletion and hf01 RTFlagPolyA reverse.

All constructs were verified and/or analyzed by DNA sequencing. Allconstructs and mutagenic libraries contained T7 TMV promoter at the 5′flanking region and Flag tag or His₆ tag sequences at 3′ flanking regionfor RNA-protein fusion production and purification in vitro.

RNA-Protein Fusion Production

For each round of selection PCR DNA was transcribed using MegaScripttranscription kit (Ambion) at 37° C. for 4 hours. Template DNA wasremoved by DNase I (Ambion) digestion at 37° C. for 20 minutes. RNA waspurified by phenol/chloroform extraction followed by gel filtration on aNAP-25 column (Amersham). The puromycin linker PEG 6/10 (5′ Pso u agcgga ugc XXX XXX CC Pu 3′, where Pso=C6-Psoralen, u,a,g,c=2′OMe-RNA,C=standard amidities, X: Spacer Phosphoramidite 9(9-O-Dimethoxytrityl-triethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite);Pu=Puromycin-CPG) was synthesized as described previously (Kurz et al,Nuc. Acid Res. 28:83, 2000). The linker was annealed to the library RNAin 0.1 M NaCl, 25 mM Tris HCl, pH 7.0, by gradient temperature decreasefrom 85° C. to 4° C. The linker and RNA were then cross linked byexposing to UV light (365 nm) for 15 minutes. The cross-linked mixture(600 pmol RNA) was included in an in vitro translation reaction usingrabbit reticulocyte lysate translation kit (Ambion) in the presence of³⁵S-labeled methionine at 30° C. for 60 minutes. To enhance fusionformation, 0.5 M KCl and 0.05 M MgCl₂ were added to the reaction andincubated for 30 minutes at 4° C. Fusion molecules were purified usingoligo-dT cellulose (Sigma) chromatography as follows. The translationand fusion mix was diluted into buffer A (100 mM Tris HCl, 1M NaCl,0.05% Tween-20, pH 8.0) and added to oligo dT cellulose. The slurry wasrotated at 4° C. for 1 hour and transferred to a spin column. Oligo dTcellulose beads were washed on the column with 10 column volumes ofbuffer A and eluted with 3 column volumes of H₂O. Reverse transcriptionreaction was conducted with SuperScript II Reverse Transcription kit(Invitrogen) for 1 hour at 42° C. using primer Hu3′FLAGSTOP. To decreasepotential non-specific binding through reactive cysteines the thiolgroups were reacted with 1 mM of 2-nitro-5-thiocyanatobenzoic acid(NTCB) or N-ethylmaleimide (NEM) alternatively over the course of theselection. The reaction was carried out for 1 hour at room temperature.Fusion molecules were further purified by anti-FLAG affinitychromatography using M2 agarose (Sigma). The M2 beads were added to thereaction and rotated in buffer B (1×PBS, 0.02% Triton X100) for 1 hourat 4° C. Then the beads were applied to a spin column, washed with 5column volumes of buffer B and fusion molecules were eluted with 3column volumes of 100 μM Flag peptide DYKDDDDK (Sigma) in buffer G.Fusion yield was calculated based on specific activity measured byscintillation counting of ³⁵S-methionine in the samples.

For the 159 (Q8L) randomized library, RNA-protein fusion was preparedusing a streamlined, semi-automated procedure in a Kingfisher™(ThermoLabSystems). The steps were similar to the procedure describedabove except for several steps described below. Purification of theRNA-protein fusion molecules was performed in buffer C (100 mM Tris HCl,60 mM EDTA, 1M NaCl, 0.05% Triton X100, pH 8.0) on magnetic oligo dTbeads (GenoVision). The beads were washed with 10 reaction volumes ofbuffer Ca (100 mM Tris HCl, 1M NaCl, 0.05% Triton X100, pH 8.0) andfusion proteins were eluted with one volume of H₂O. Reversetranscription (RT) was conducted using primer C2RT. Fusion proteins werefurther purified by His-tag affinity chromatography using Ni-NTAmagnetic beads (Qiagen). The RT reaction was incubated with Ni-NTA beadsin buffer D (2M NaCl, 0.05% Triton) for 20 minutes at room temperature,the beads were then washed with 10 reaction volumes of buffer E (1×PBS,0.05% Triton X100, pH 7.4) and fusion molecules were eluted with onevolume of buffer F (1×PBS, 0.05% Triton X100, 100 mM imidazole, pH 7.4).

Selection:

Primary selection against KDR. Fusion library (˜10¹³ clones in 1 ml) wasincubated with 150 μl of Protein A beads (Dynal) which waspre-immobilized with 200 nM of human IgG1 for 1 hour at 30° C. prior toselection to reduce non-specific binding to both Protein A beads and Fcprotein (preclear). The supernatant was then incubated in buffer G (50mM HEPES, 150 mM NaCl, 0.02% Triton X-100, 1 mg/ml bovine serum albumin,0.1 mg/ml salmon sperm DNA, pH 7.4) with KDR-Fc chimera for 1 hour at30° C. with end-over-end rotation. Final concentrations of KDR-Fc were250 nM for Round 1, 100 nM for rounds 2-4 and 10 nM for rounds 5 and 6.The target was captured on 300 μl of Protein A beads (Round 1) or 100 μlof Protein A beads (Rounds 2-6) for 30 minutes at 30° C. withend-over-end rotation and beads were washed 5 times with 1 ml of bufferG (50 mM HEPES, 150 mM NaCl, 0.02% Triton X-100, pH 7.4). Bound fusionmolecules were eluted with 100 μl of 0.1 M KOH into 50 μl of 1 M TrisHCl, pH 8.0. DNA was amplified from elution by PCR using flankingprimers T7TMV Fn and MK165-4 A20.

Affinity and specificity maturation of KDR binder VR28. Clone VR28 wasmutagenized by hypermutagenic PCR or oligo-directed mutagenesis asdescribed above and fusion sub-libraries were constructed. Followingpre-clear with Protein A beads selection was performed in buffer I(1×PBS, 0.02% Triton X-100, 1 mg/ml bovine serum albumin, 0.1 mg/mlsalmon sperm DNA, pH 7.4) for four rounds according to proceduredescribed above. DNA was amplified from elution by PCR using primersT7TMV Fn and MK165-4 A20. Lower target concentrations (0.1 nM KDR forfirst four rounds of selection) were used for libraries derived fromoligo mutagenesis and then 1 nM mouse VEGF-R2 (Flk-1) was introduced forthree additional rounds of selection. Primers T7 TMV N-terminus deletionand MK165-4 A20 were used for PCR in the last 3 rounds.

For specificity maturation of KDR binder 159 first 6 positions of the FGloop of clone 159 Q(8)L were randomized by PCR as described above.Binding of the fusion sub-library to biotinylated mouse VEGF-R2 (70 nM)was performed in buffer I at room temperature for 30 minutes. The restof the selection procedure was continued in Kingfisher™(ThermoLabSystems). The biotinylated target was captured on 50 μl ofstreptavidin-coated magnetic beads (Dynal) and the beads were washedwith 10 volumes of buffer I and one volume of buffer J (1×PBS, 0.02%Triton X-100, pH 7.4). Bound fusion molecules were eluted with 100 μl of0.1 M KOH into 50 μl of 1 M Tris HCl, pH 8.0. DNA was amplified fromelution by PCR using primers F1U2 and C2asaiA20. After four rounds ofselection an off-rate/rebinding selection against 7 nM Flk-1 was appliedfor another two rounds as follows. After the binding reaction withbiotinylated mouse Flk-1 had progressed for 30 minutes, a 100-foldexcess of non-biotinylated Flk-1 was added and the reaction continuedfor another 6 hours to allow time for the weak binders to dissociate.The biotinylated target was captured on 50 μl of streptavidin beads(Dynal) and beads were washed 5 times with 1 ml of buffer J. Boundfusion molecules were eluted by incubation at 75° C. for 5 minutes.Supernatant was subjected to re-binding to 7 nM Flk-1 and standardselection procedure was continued. DNA from the final elution pool wassubjected to DE loop randomization (see above) and fusion sub-librarywas selected against 7 nM mouse VEGF-R2 for three rounds. At the fourthround an off-rate selection was applied with re-binding to 1 nM humanVEGF-R2. Final DNA was amplified from elution by PCR using primers F1U2and C2asaiA20.

Radioactive Equilibrium Binding Assay

To prepare ³⁵S-labeled binding proteins for analysis, mRNA was preparedas described above for RNA-protein fusion production but the linkerligation step was omitted. The mRNA was expressed using rabbitreticulocyte lysate translation kit (Ambion) in the presence of³⁵S-labeled Met at 30° C. for 1 hour. Expressed protein was purified onM2-agarose Flag beads (Sigma) as described above. This procedureproduced the encoded protein without the nucleic acid tail. In a directbinding assay, VEGF-R2-Fc fusions in concentrations ranging from 0 to200 nM were added to a constant concentration of the purified protein(0.2 or 0.5 nM) and incubated at 30° C. for 1 hour in buffer B. Thereceptor-binder complexes were captured using Protein A magnetic beadsfor another 10 minutes at room temperature using a Kingfisher™. Thebeads were washed with six reaction volumes of buffer B. The protein waseluted from the beads with 100 μL of 0.1 M KOH. 50 μL of the reactionmixture and elution were dried onto a LumaPlate-96 (Packard) and theamount of ³⁵S on the plate was measured using a TopCount NXT instrument(Packard). The amount of fibronectin-based scaffold protein bound to thetarget was estimated as a percent of radioactivity eluted from Protein Amagnetic beads compare to radioactivity in the reaction mixture.Nonspecific binding of fibronectin-based scaffold proteins to the beadswas determined by measuring binding in the absence of KDR-Fc andrepresented less than 1-2% of the input. Specific binding was obtainedthrough subtraction of nonspecific binding from total binding. Data wasanalyzed using the GraphPad Prizm software (GraphPad Software, Inc, SanDiego, Calif.), fitted using a one site, non-linear binding equation.

Expression and Purification of Soluble Fibronectin-Based ScaffoldProtein Binders:

For expression in E. coli residues 1-101 of each clone followed by theHis₆ tag were cloned into a pET9d-derived vector and expressed in E.coli BL21 (DE3) pLysS cells (Invitrogen). 20 ml of overnight culture wasused to inoculate 1 liter of LB medium containing 50 μg/mL kanamycin and34 μg/mL chloramphenicol. The culture was grown at 37° C. until A₆₀₀0.4-0.6. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG,Invitrogen) the culture was grown for another 3 hours at 37° C. andharvested by centrifugation for 30 minutes at 3,000 g at 4° C. The cellpellet was resuspended in 50 mL of lysis buffer K (50 mM NaH₂PO₄, 0.5 MNaCl, 5% glycerol, 5 mM CHAPS, 25 mM imidazole, 1× Complete™ ProteaseInhibitor Cocktail (Roche), pH 8.0) BufferL and sonicated on ice at 80 Wfor four 15 second pulses separated by ten-second pauses. The solublefraction was separated by centrifugation for 30 minutes at 30,000 g at4° C. The supernatant was rotated for 1 hour at 4° C. with 10 mL ofTALON™ SuperflowTM Metal Affinity Resin (Clontech) pre-equilibrated withwash buffer L (50 mM NaH₂PO₄, 0.5 M NaCl, 5% glycerol, 25 mM imidazole,pH 8.0). The resin was then washed with 10 column volumes of buffer Land 30 column volumes of buffer M (1×PBS, pH 7.4, 25 mM imidazole, pH7.4). Protein was eluted with 5 column volumes of buffer N (1×PBS, 250mM imidazole, pH 7.4) and dialyzed against 1×PBS at 4° C. Anyprecipitate was removed by filtering at 0.22 μm (Millipore).

BIAcore Analysis of the Soluble Fibronectin-Based Scaffold Proteins:

The binding kinetics of fibronectin-based scaffold proteins bindingproteins to the target was measured using BIAcore 2000 biosensor(Pharmacia Biosensor). Human and mouse VEGF-R2-Fc fusions wereimmobilized onto a CM5 sensor chip and soluble binding proteins wereinjected at concentrations ranging from 0 to 100 nM in buffer 0 (10 mMHEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4). Sensorgrams were obtainedat each concentration and were evaluated using a program, BIA Evaluation2.0 (BIAcore), to determine the rate constants k_(a) (k_(on)) and k_(d)(k_(off)). The affinity constant, K_(D) was calculated from the ratio ofrate constants k_(off)/k_(on).

For inhibition experiments, human VEGF₁₆₅ was immobilized on a surfaceof CM-5 chip and KDR-Fc was injected at a concentration of 20 nM in thepresence of different concentrations of soluble binding proteins rangingfrom 0 to 100 nM. IC₅₀ was determined at a concentration when only 50%of KDR-Fc binding to the chip was observed.

Reversible Refolding of A VEGFR Binding Polypeptide:

Differential scanning calorimetry (DSC) analysis was performed on M5FLprotein in 100 mM sodium acetate buffer (pH 4.5). An initial DSC run(Scan 1) was performed in a N-DSC II calorimeter (Calorimetry SciencesCorp) by ramping the temperature from 5-95° C. at a rate of 1 degree perminute, followed by a reverse scan (not shown) back to 10 degrees,followed by a second run (Scan 2). Under these conditions, data werebest fit using a two transition model (Tm=77° C. and 67° C. using Orginsoftware (OrginLab Corp)). See FIG. 10.

PEGylation of the M5FL Protein:

The C100-form of the M5FL protein, which has the complete sequence ofM5FL with the Ser at position 100 mutated to a Cysteine including theadditional C-terminal His-tag used to purify the protein. The purifiedM5FL-C100 protein was modified at the single cysteine residue byconjugating various maleimide-derivatized PEG forms (Shearwater). Theresulting reacted proteins were run on a 4-12% polyacrylamide gel (FIG.11).

Construction of Cell Lines:

Plasmid construction. Plasmids, encoding chimeric receptors composed ofthe transmembrane and cytoplasmic domains of the human erythropoietinreceptor (EpoR) fused to the extracellular domains of KDR, Flk-1, orhuman TrkB were constructed by a two-step PCR procedure. PCR productsencoding the extracellular domains were amplified from plasmids encodingthe entire receptor gene: KDR (amino acids 1 to 764) was derived fromclone PR1371_H11 (OriGene Technologies, Rockville, Md.) with primers5-RI-hKDR-1B/3-EPO/hKDR-2312B, flk-1 (amino acids 1 to 762) was derivedfrom clone #4238984 (IMAGE) with primers 5-RI-mKDR-1/3-EPO/mKDR-2312,and human TrkB (from amino acids 1 to 430) from clone #X75958(Invitrogen Genestorm) with primers 5-RI-hTrkB-1/3-EpoR/hTrkB-1310. PCRproducts encoding the EpoR transmembrane and cytoplasmic domains (aminoacids 251 to 508) were amplified from clone #M60459 (InvitrogenGenestorm) with the common primer 3-XHO-EpoR-3066 and one of threegene-specific primers 5-hKDR/EPO-2274B (KDR), 5-mKDR/EPO-2274 (flk-1),and 5′hTrkB/EpoR-1274 (human TrkB), which added a short sequencecomplementary to the end of the receptor fragment PCR product. Second,PCR products encoding the two halves of the chimeric genes were mixedand amplified with 3-XHO-EpoR-3066 and the 5′ primers (5-RI-hKDR-1B,5-RI-mKDR-1, and 5-RI-hTrkB-1) specific for each gene used in theprevious cycle of amplification. The resulting PCR products weredigested with EcoRI and XhoI and cloned into pcDNA3.1 (+) (Invitrogen)to generate the plasmids phKE8 (human KDR/EpoR fusion), pmKE2(flk-1/EpoR fusion), and phTE (TrkB/EpoR fusion).

Construction of cell lines for flow cytometry. CHO-K1 cells (AmericanType Culture Collection, Manassas, Va.) were stably transfected usingLipofectamine 2000 (Invitrogen) with either pcDNA 3.1 (Invitrogen)alone, pmKE2 alone, or a mixture of pcDNA 3.1 and a plasmid encodingfull-length human KDR (Origene Inc., clone PR1371-H11). Stabletransfectants were selected and maintained in the presence of 0.5 mg/mlof Geneticin (Invitrogen). The human KDR-expressing clone designatedCHO-KDR and the murine VEGFR-2/EpoR-chimera-expressing populationdesignated CHO-Flk were obtained by fluorescence activated cell sortingof the transfected population following staining with an anti-KDRpolyclonal antiserum (R&D Systems). CHO-KDR and CHO-Flk cell lines weregrown routinely in Dulbecco's modified Eagle's medium (DMEM; Gibco)supplemented with 10% (v/v) fetal bovine serum (FBS), 0.5 mg/mlGeneticin, 100 U/ml penicillin, 0.25 μg/ml amphotericin B, 100 μg/mlstreptomycin and 2 mM L-glutamine.

Construction of Ba/F3 cell lines. Cell lines that would proliferate inresponse to VEGF binding by VEGFR-2 were constructed by transfection ofthe murine pre-B cell line Ba/F3 (DSMZ, Braunschweig, Germany) withphKE8 or pmKE2, receptor chimeras consisting of the extracellulardomains of human or murine VEGFR-2 fused to the transmembrane andcytoplasmic domains of the human erythropoietin receptor (see above).Ba/F3 cells were maintained in minimal Ba/F3 medium (RPMI-1640 (Gibco)containing 10% FBS, 100 U/ml penicillin, 0.25 μg/ml amphotericin B, 100μg/ml streptomycin and 2 mM L-glutamine) supplemented with 10%conditioned medium from WEHI-3B cells (DSMZ; grown in Iscove's modifiedDulbecco's medium (Gibco)/10% FBS/25 μM β-mercaptoethanol) as a sourceof essential growth factors. Following electroporation with the plasmidspmKE2 or phKE8, stable transfectants were selected in 0.75 mg/mlGeneticin. Geneticin-resistant populations were transferred to minimalBa/F3 medium containing 100 ng/ml of human VEGF₁₆₅ (R&D Systems), andthe resulting VEGF-dependent populations were designated Ba/F3-Flk andBa/F3-KDR. Control cell line expressing a chimeric TrkB receptor(Ba/F3-TrkB) that would be responsive to stimulation by NT-4, thenatural ligand for TrkB was similarly constructed using the plasmid phTEand human NT-4 (R&D Systems).

Analysis of Cell Surface Binding of Fibronectin-Based Scaffold Proteins:

Binding of fibronectin-based scaffold protein to cell-surface KDR andFlk-1 was analyzed simultaneously on VEGF-R2-expressing and controlcells by flow cytometry. CHO-pcDNA3 cells (control) were released fromtheir dishes with trypsin-EDTA, washed in Dulbecco's PBS without calciumand magnesium (D-PBS⁻; Invitrogen), and stained for 30 minutes at 37° C.with 1.5 μM CMTMR(5-(and-6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine)(Molecular Probes). The cells were washed in D-PBS⁻ and incubated for afurther 30 minutes at 37° C., and then resuspended in blocking buffer(D-PBS⁻/10% fetal bovine serum) on ice. CHO-KDR or CHO-Flk cells weretreated identically except that CMTMR was omitted. 75,000 ofCMTMR-stained CHO-pcDNA3 cells were mixed with an equal number ofunstained CHO-KDR or CHO-Flk cells in each well of a V-bottom 96-wellplate. All antibodies and fibronectin-based scaffold proteins werediluted in 25 μl/well of blocking buffer, and each treatment wasconducted for 1 hour at 4° C. Cell mixtures were stained withHis₆-tagged fibronectin-based scaffold proteins, washed twice with coldD-PBS⁻, and then treated with 2.5 μg/ml anti-His₆ MAb (R&D Systems),washed, and stained with 4 μg/ml Alexa Fluor 488-conjugated anti-mouseantibody (Molecular Probes). For cells treated with an anti-KDR mousemonoclonal antibody (Accurate Chemical, Westbury, N.Y.) or an anti-flk-1goat polyclonal antibody (R&D Systems), the anti-His₆ step was omitted,and antibody binding was detected with the species-appropriate AlexaFluor 488 conjugated secondary antibody (Molecular Probes). Followingstaining, cells were resuspended in 200 μl/well D-PBS⁻/1% FBS/1 μg/ml7-aminoactinomycin D (7-AAD; Molecular Probes) and analyzed by flowcytometry on a FACSCalibur (Becton Dickinson, San Jose, Calif.) equippedwith a 488 nM laser. Following gating to exclude dead cells (7-AADpositive), VEGFR-2-expressing cells and CHO-pcDNA3 cells were measuredindependently for Alexa Fluor 488 fluorescence by gating on theCMTMR-negative or -positive populations, respectively. Controlexperiments showed that staining with CMTMR did not interfere with thedetection of Alexa Fluor 488-conjugated antibodies on the surface of thestained cells.

Cell-surface binding was also assessed by fluorescence microscopy usingthe secondary antibodies described above. For these studies, antibodieswere diluted in D-PBS containing calcium and magnesium (D-PBS⁺)/10% FBS.Cells were grown on 24- or 96-well plates, and following staining werekept in D-PBS⁺ for observation on an inverted fluorescence microscope.

Ba/F3 Cell Proliferation Assay:

Ba/F3 cells were washed three times in minimal Ba/F3 medium andresuspended in the same medium containing 15.8 ng/ml of proliferationfactor (human VEGF₁₆₅, murine VEGF₁₆₄, or hNT-4 for Ba/F3-KDR,Ba/F3-Flk, or Ba/F3-TrkB cells, respectively), and 95 μl containing5×10⁴ Ba/F3-KDR cells or 2×10⁴ Ba/F3-Flk or Ba/F3-TrkB cells were addedper well to a 96-well tissue culture plate. 5 μl of serial dilutions oftest protein in PBS was added to each well for a final volume of 100 μlBa/F3 medium/5% PBS/15 ng/ml growth factor. After incubation for 72hours at 37° C., proliferation was measured by addition of 20 μl ofCellTiter 96® Aqueous One Solution Reagent (Promega) to each well,incubation for 4 hours at 37° C., and measurement of the absorbance at490 nm using a microtiter plate reader (Molecular Dynamics).

HUVEC Cell Proliferation Assay:

HUVEC cells (Clonetics, Walkersville, Md.) from passage 2-6 were grownin EGM-2 medium (Clonetics). 5000 cells/well were resuspended in 200 μlstarvation medium (equal volumes of DMEM (Gibco) and F-12K medium(ATCC), supplemented with 0.2% fetal bovine serum and 1×penicillin/streptomycin/fungizone solution (Gibco)), plated in 96-welltissue culture plates and incubated for 48 hours. Fibronectin-basedbinding proteins were added to the wells and incubated for 1 hour at37°, and then human VEGF₁₆₅ was added to a final concentration of 16ng/ml. After 48 hours incubation, cell viability was measured byaddition of 30 μl/well of a mixture of 1.9 mg/ml CellTiter96® AQueousMTS reagent (Promega) with 44 μg/ml phenazine methosulfate (Sigma) andmeasurement of absorbance at 490 nm as described above for Ba/F3 cells.

Example 12 Antibody Light Chain-Based VEGFR Binding Polypeptides

FIGS. 21A and 21B show amino acid sequences of VEGFR bindingpolypeptides (SEQ ID NOs:241-310) based on an antibody light chainvariable region (VL) framework/scaffold.

Light chain variable domain proteins were generated using the PROfusion™system, as described above for use with ¹⁰Fn3-derived proteins.

All references cited herein are hereby incorporated by reference intheir entirety. TABLE 1 Preferred Specific Peptide Sequences N- BindingBinding Kd SEQ Clone ter- BC DE to 1 nM Kd KDR, to 1 nM FLK, ID NO Nameminus Loop Loop FG Loop KDR, % nM FLK, % nM KDR Binders 6 K1 Del 1-8RHPHFPTR LQPPT M G L Y G H E L L T P 48 0.55 7 K2 Del 1-8 RHPHFPTR LQPPTD G E N G Q F L L V P 48 1.19 8 K5 Del 1-8 RHPHFPTR LQPPT M G P N D N EL L T P 47 1.54 9 K3 Del 1-8 RHPHFPTR LQPPT A G W D D H E L F I P 451.15 10 K7 Del 1-8 RHPHFPTR LQPPT S G H N D H M L M I P 40 2.2 11 K4 Del1-8 RHPHFPTR LQPPT A G Y N D Q I L M T P 38 1.95 12 K9 Del 1-8 RHPHFPTRLQPPT F G L Y G K E L L I P 35 1.8 13 K10 Del 1-8 RHPHFPTR LQPPT T G P ND R L L F V P 33 0.57 14 K12 Del 1-8 RHPHFPTR LQPPT D V Y N D H E I K TP 29 0.62 15 K6 Del 1-8 RHPHFPTR LQPPT D G K D G R V L L T P 27 0.93 16K15 Del 1-8 RHPHFPTR LQPPT E V H H D R E I K T P 25 0.35 17 K11 Del 1-8RHPHFPTR LQPPT Q A P N D R V L Y T P 24 1.16 18 K14 Del 1-8 RHPHFPTRLQPPT R E E N D H E L L I P 20 0.57 19 K8 Del 1-8 RHPHFPTR LQPPT V T H NG H P L M T P 18 3.3 20 K13 Del 1-8 RHPHFPTR LQPPT L A L K G H E L L T P17 0.58 21 VR28 WT RHPHFPTR LQPPT V A Q N D H E L I T P 3 11 22 159 WTRHPHFPTR LQPPA M A Q S G H E L F T P KDR and FLK Binders 24 E29 Del 1-8RHPHFPTR LQPPT V E R N G R V L M T P 41 44 1.51 0.91 25 E19 Del 1-8RHPHFPTR LQPPT V E R N G R H L M T P 38 40 1.3 0.66 33 E25 Del 1-8RHPHFPTR LQPPT L E R N G R E L M T P 41 28 1.58 1.3 45 E9 Del 1-8RHPHFPTR LQPPT E E R N G R T L R T P 24 34 2.37 1.4 50 E24 Del 1-8RHPHFPTR LQPPT V E R N D R V L F T P 24 29 54 E26 Del 1-8 RHPHFPTR LQPPTV E R N G R E L M T P 27 20 1.66 2.05 59 E28 Del 1-8 RHPHFPTR LQPPT L ER N G R E L M V P 19 21 1.63 2.1 60 E3 Del 1-8 RHPHFPTR LQPPT D G R N DR K L M V P 37 14 0.96 5.4 65 E5 Del 1-8 RHPHFPTR LQPPT D G Q N G R L LN V P 26 10 0.4 3.2 91 E23 Del 1-8 RHHPHFPTR LQPPT V H W N G R E L M T P36 7 92 E8 Del 1-8 RHPHFPTR LQPPT E E W N G R V L M T P 51 10 93 E27 Del1-8 RHPHFPTR LQPPT V E R N G H T L M T P 37 9 94 E16 Del 1-8 RHPHFPTRLQPPT V E E N G R Q L M T P 35 0 95 E14 Del 1-8 RHPHFPTR LQPPT L E R N GQ V L F T P 33 11 96 E20 Del 1-8 RHPHFPTR LQPPT V E R N G Q V L Y T P 4311 97 E21 Del 1-8 RHPHFPTR LQPPT W G Y K D H E L L I P 47 1 98 E22 Del1-8 RHPHFPTR LQPPT L G R N D R E L L T P 45 3 99 E2 Del 1-8 RHPHFPTRLQPPT D G P N D R L L N I P 53 10 100 E12 Del 1-8 RHPHFPTR LQPPT F A R DG H E I L T P 36 1 101 E13 Del 1-8 RHPHFPTR LQPPT L E Q N G R E L M T P38 1 102 E17 Del 1-8 RHPHFPTR LQPPT V E E N G R V L N T P 32 10 103 E15Del 1-8 RHPHFPTR LQPPT L E P N G R Y L M V P 52 2 104 E10 Del 1-8RHPHFPTR LQPPT E G R N G R E L F I P 53 3 154 M2 WT RHPHFPTR LQPPA W E RN G R E L F T P 156 M3 WT RHPHFPTR LQPPA K E R N G R E L F T P 172 M4 WTRHPHFPTH LQPPA T E R T G R E L F T P 173 M8 WT RHPHFPTH LQPPA K E R S GR E L F T P 175 M6 WT RHPHFPTH LQPPA L E R D G R E L F T P 180 M7 WTRHPHFPTR LQPTT W E R N G R E L F T P 181 M1 WT RHPHFPTR LQPTV L E R N DR E L F T P 177 M5FL WT RHPHFPTR LQPPL K E R N G R E L F T P

TABLE 2 KDR & FLK binders Bind- Bind- ing ing to 1 to 1 SEQ nM nM Kd KdID Clone N- N-Terminus DE Frame- KDR, Flk-1, KDR, Flk, NO Name terminusFramework 1 BC Loop Framework 2 Loop Framework 3 FG Loop work 4 % % nMnM  23 D12 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPTATISGLKPGVDYTITGYAVT V E R N G R K L M T P ISINYRT 46 47  24 E29 Del 1-8EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVTV E R N G R V L M T P ISINYRT 41 44 1.51 0.91  25 E19 Del 1-8EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVTV E R N G R H L M T P ISINYRT 38 40 1.3 0.66  26 D1 Del 1-8EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVTV E R N G R M L M T P ISINYRT 38 38  27 C6 Del 1-8 EVVAATPTSLLISWRHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R VL M T P ISINYRT 36 49  28 EGE5 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R V L N T PISINYRT 32 47  29 EGE2 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R Q L M T PISINYRT 42 33  30 D4 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R T L F T PISINYRT 27 44  31 E25 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R E L M T PISINYRT 41 28 1.58 1.3  32 EGE6 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R L L N T PISINYRT 33 40  33 C7 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT H E R N G R V L M T PISINYRT 32 40  34 D9 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT E E R N G R V L F T PISINYRT 31 40  35 EGE3 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R Q L Y T PISINYRT 34 38  36 D3 Del 1-8 EVVAATPTSLUSW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R A L M T PISINYRT 36 30  37 D2 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R N L M T PISINYRT 35 30  38 C8 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R V L I T PISINYRT 30 34  39 EGE4 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R V L N T PISINYRT 26 41  40 D7 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G K V L M T PISINYRT 39 27  41 D5 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R T L M M PISINYRT 38 23  42 B3 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT M E R N G R E L M T PISINYRT 33 27  43 E9 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT E E R N G R T L R T PISINYRT 24 34 2.37 1.4  44 D6 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G K T L M T PISINYRT 32 30  45 EGE7 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N D R V L L T PISINYRT 31 30  46 EGE1 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R K L M T PISINYRT 30 29  47 F9 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E P N G R V L N T PISINYRT 32 23  48 E24 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N D R V L F T PISINYRT 24 29  49 B11 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R E L K T PISINYRT 29 21  50 B12 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R E L R T PISINYRT 29 21  51 B5 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT Q E R N G R E L M T PISINYRT 27 24  52 E26 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R E L M T PISINYRT 27 20 1.66 2.05  53 C12 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R V L S V PISINYRT 24 20  54 F4 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R D G R T L R T PISINYRT 31 18  55 E18 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R E L N T PISINYRT 17 29 1.2 0.53  56 C11 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R V L I V PISINYRT 19 21  57 E28 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R E L M V PISINYRT 19 21 1.63 2.1  58 E3 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G R N D R K L M V PISINYRT 37 14 0.96 5.4  59 F8 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E H N G R T S F T PISINYRT 33 13  60 F3 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R D G R K L Y T PISINYRT 27 15  61 B10 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R E L N T PISINYRT 15 23  62 E6 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G W N G R L L S I PISINYRT 36 7 0.35 7.1  63 E5 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G Q N G R L L N V PISINYRT 26 10 0.4 3.2  64 G4 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT I E K N G R H L N I PISINYRT 21 12  65 A3 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G W N G K M L S V PISINYRT 33 7  66 A4 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G V N D R L L F I PISINYRT 46 2  67 A6 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G P N D R L L N I PISINYRT 18 2  68 A7 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G P N N R E L I V PISINYRT 18 2  69 A8 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G L N G K Y L F V PISINYRT 38 4  70 A9 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT E G W N D R E L F V PISINYRT 31 4  71 A10 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT F G W N G R E L L T PISINYRT 34 4  72 EGE11 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT F G W N D R E L L I PISINYRT 50 0  73 A11 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E W N N R V L M T PISINYRT 26 6  74 A12 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E W N G R V L M T PISINYRT 40 10  75 B4 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT N E R N G R E L M T PISINYRT 19 12  76 B6 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G K E L M T PISINYRT 23 11  77 B7, Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R E L L T PISINYRT 18 10 B8  78 B11 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R E L K T PISINYRT 29 21  79 C1 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT Q E R N G R E L R T PISINYRT 28 13  80 C2 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R E L L W PISINYRT 40 16  81 C3 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G R E L M I PISINYRT 31 17  82 C9 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G L V L M T PISINYRT 33 7  83 C10 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G R V L I I PISINYRT 24 17  84 D11 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G H K L F T PISINYRT 24 3  85 EGE8 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N E R V L M T PISINYRT 26 20  86 EGE9 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT F G P N D R E L L T PISINYRT 32 1  87 EGE10 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT M G P N D R E L L T PISINYRT 37 1  88 EGE11 Del 1-8 EVVAATPTSLLISW RHHPHFPTYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT M G K N D R E L L T PISINYRT 32 1 R  89 E23 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V H W N G R E L M T PISINYRT 36 7  90 E8 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT E E W N G R V L M T PISINYRT 51 10  91 E27 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G H T L M T PISINYRT 37 9  92 E16 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E E N G R Q L M T PISINYRT 35 0  93 E14 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N G Q V L F T PISINYRT 33 11  94 E20 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R N G Q V L Y T PISINYRT 43 11  95 E21 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT W G Y K D H E L L I PISINYRT 47 1  96 E22 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L G R N D R E L L T PISINYRT 45 3  97 E2 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G P N D R L L N I PISINYRT 53 10  98 E12 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT F A R D G H E I L T PISINYRT 36 1  99 E13 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E Q N G R E L M T PISINYRT 38 1 100 E17 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E E N G R V L N T PISINYRT 32 10 101 E15 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E P N G R V L M V PISINYRT 52 2 102 E10 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT E G R N G R E L F I PISINYRT 53 3 103 F1 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT S G R N D R E L L V PISINYRT 18 2 104 F5 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E R D G R E L N I PISINYRT 12 8 105 F6 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E Q N G R V L M T PISINYRT 37 2 106 F7 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E H N G R V L N I PISINYRT 30 7 107 F10 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT M A P N G R E L L T PISINYRT 29 1 108 F11 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT V E Q N G R V L N T PISINYRT 20 8 109 F12 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT D G R N G H E L M T PISINYRT 17 1 110 G1 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT E G R N G R E L M V PISINYRT 22 2 111 G2 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT L E R N N R E L L T PISINYRT 25 9 112 G3 Del 1-8 EVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPT ATISGLKPGVDYTITGYAVT M E R S G R E L M T PISINYRT 28 10 113 MWF10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT R A L L S I E L F T PISINYRT 114 MWA10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT F A R K G T E L F T PISINYRT 115 MWA2 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT L E R C G R E L F T PISINYRT 116 MWC10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT R E R N G R E L F T PISINYRT 117 MWB7 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT K E R N G R E L F T PISINYRT 118 MWH8 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT C E R N G R E L F T PISINYRT 119 MWA10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT L E R T G R E L F T PISINYRT 120 MWB2 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W E R T G K E L F T PISINYRT 121 MWC3- WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT I E R T C R E L F T PISINYRT f1 122 MWG11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT G G M I V R E L F T PISINYRT 123 MWG11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT F G R S S R E L F T PISINYRT 124 MWD3- WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT R H K S R G E L F T PISINYRT f1 125 MWE11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT R H R D K R E L F T PISINYRT 126 MWD10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT Y H R G R G E L F T PISINYRT 127 MWC1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT R H R G C R E L F T PISINYRT 128 MWA12 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT S H R L R K E L F T PISINYRT 129 MWB3- WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT M H R Q R G E L F T PISINYRT f1 130 MWA11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT F H R R R G E L F T PISINYRT 131 MWG12 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT F H R R R G E L F T PISINYRT 132 MWH11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT S H R R R N E L F T PISINYRT 133 MWD12 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT L H R R V R E L F T PISINYRT 134 MWH5 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT R H R R R G E L F T PISINYRT 135 MWA1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W H R S R K E L F T PISINYRT 136 MWG4- WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT R H R S R G E L F T PISINYRT f1 137 MWA12 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT V H R T G R E L F T PISINYRT 138 MWG11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W H R V R G E L F T PISINYRT 139 MWC12 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W H R V R G E L F T PISINYRT 140 MWF11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W H R W R G E L F T PISINYRT 141 MWE11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W K R S G G E L F T PISINYRT 142 MWD10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT R L X N X V E L F T PISINYRT 143 MWC4- WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W R T P H A E L F T PISINYRT f1 144 MWF3 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT L S P H S V E L F T PISINYRT 145 MWB2 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT V S R Q K A E L F T PISINYRT 146 MWE10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT S S Y S K L E L F T PISINYRT 147 MWD9 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT L T D R G S E L F T PISINYRT 148 MWH3- WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT G T R T R S E L F T PISINYRT f1 149 MWG10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT P V A G C S E L F T PISINYRT 150 MWH11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W W Q T P R E L F T PISINYRT 151 MWF11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTRYYRITYGETGGNSPVQEFTVP LQPPA ATISGLKPGVDYTITGYAVT W W Q T P R E L F T PISINYRT 152 M2 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVPLQPPA ATISGLKPGVDYTITGYAVT W E R N G R E L F T P ISINYRT 153 MWB09- WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT W E W N G R E L F T P ISINYRT f1 154 M3 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT K E R N G R E L F T P ISINYRT 155 MWA3 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT G A L N T S E L F T P ISINYRT 156 MWE10 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT F G R E R R E L F T P ISINYRT 157 MWG33 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT S G R V S F E L F T P ISINYRT 158 MWD5 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT F H R R R G E L F T P ISINYRT 159 MWC3 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT L I R M N T E L F T P ISINYRT 160 MWH3 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT C L H L I T E L F T P ISINYRT 161 MWC2 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT V L K L T L E L F T P ISINYRT 162 MWE2 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT V L K L T L E L F T P ISINYRT 163 MWA2 WTVSDVPRDLEVVAATPTSLLISW FHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT V L K L T L E L F T P ISINYRT 164 MWD3 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT A L M A S G E L F T P ISINYRT 165 MWE3 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT S M K N R L E L F T P ISINYRT 166 MWB3 WTVSDVPRDLEVVAATPTSLLISW FHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT L R C L I P E L F T P ISINYRT 167 MWD2 WTVSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT V S R Q K A E L F T P ISINYRT 168 MWC11 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT W S R T G R E L F T P ISINYRT 169 MWH12 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT V W R T G R E L F T P ISINYRT 170 M4 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT T E R T G R E L F T P ISINYRT 171 M8 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT K E R S G R E L F T P ISINYRT 172 MWF10- WTVSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT L E R N D R E L F T P ISINYRT f1 173 M6 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPAATISGLKPGVDYTITGYAVT L E R D G R E L F T P ISINYRT J 174 MWB6 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPTATISGLKPGVDYTITGYAVT Q G R H K R E L F T P ISINYRT 175 M5FL WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPLATISGLKPGVDYTITGYAVT K E R N G R E L F T P ISINYRT 176 MWG10- WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPIATISGLKPGVDYTITGYAVT M A Q N D H E L I T P ISINYRT f1 177 WD08- WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPIATISGLKPGVDYTITGYAVT M A Q N D H E L I T P ISINYRT f1; N4 178 M7 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPTTATISGLKPGVDYTITGYAVT W E R N G R E L F T P ISINYRT 179 M1 WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPTVATISGLKPGVDYTITGYAVT L E R N D R E L F T P ISINYRT 180 MWA07- WTVSDVPRDLEVVAATPTSLLISW RPPHFPTR YYRITYGETGGNSPVQEFTVP LQPTVATISGLKPGVDYTITGYAVT L E R N D R E L F T P ISINYRT f1 181 MWH11- WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP PQPPAATISGLKPGVDYTITGYAVT K E R S G R E L F T P ISINYRT f1 182 WF09- WTVSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP PQPPAATISGLKPGVDYTITGYAVT L E R N D R E L F T P ISINYRT f1; F4 183 MWG12- WTVSDVPRDLEVVAATPTSLLISW CHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPIATISGLKPGVDYTITGYAVT M A Q N D H E L I T P ISINYRT f1

TABLE 3 KDR binders N- Binding SEQ Clone ter- BC DE to 1 nM Kd KDR, IDNO Name minus Loop Loop FG Loop KDR, % nM 6 K1 Del 1-8 RHPHFPTR LQPPT MG L Y G H E L L T P 48 0.55 7 K2 Del 1-8 RHPHFPTR LQPPT D G E N G Q F LL V P 48 1.19 8 K5 Del 1-8 RHPHFPTR LQPPT M G P N D N E L L T P 47 1.549 K3 Del 1-8 RHPHFPTR LQPPT A G W D D H E L F I P 45 1.15 311 3′E9 PR4Del 1-8 RHPHFPTR LQPPT V E Q D G H V L Y I P 44 312 2′Del E6 PR4 Del 1-8RHPHFPTR LQPPT M G K N G H E L L T P 43 313 3′D3 PR4 Del 1-8 RHPHFPTRLQPPT P G P G D R E L I T P 42 314 2′Del F8 PR4 Del 1-8 RHPHFPTR LQPPT AG P G A H E L L T P 42 315 4′B3 PR4 Del 1-8 RHPHFPTR LQPPT M A Q N N R EL L T P 42 316 3′E3 PR4 Del 1-8 RHPHFPTR LQPPT M A Q Y G R E L L T P 4110 K7 Del 1-8 RHPHFPTR LQPPT S G H N D H M L M I P 40 2.2 317 3′H11 PR4Del 1-8 RHPHFPTR LQPPT L A H N G N E L L T P 39 318 3′B4 PR4 Del 1-8RHPHFPTR LQPPT V A W N G H E L M T P 38 11 K4 Del 1-8 RHPHFPTR LQPPT A GY N D Q I L M T P 38 1.95 319 2′Del F7 PR4 Del 1-8 RHPHFPTR LQPPT L G LR D R E L F V P 38 320 2′Del D3 PR4 Del 1-8 RHPHFPTR LQPPT S G L N D R VL F I P 38 321 3′C6 PR4 Del 1-8 RHPHFPTR LQPPT M G P N D R E L L T P 37322 3′F3 PR4 Del 1-8 RHPHFPTR LQPPT L G H N D R E L L T P 37 323 3′H3PR4 Del 1-8 RHPHFPTR LQPPT L G L N D R E L M T P 36 324 1′Del G10 PR4Del 1-8 RHPHFPTR LQPPT M A Q N G H K L M T P 36 12 K9 Del 1-8 RHPHFPTRLQPPT F G L Y G K E L L I P 35 1.8 325 2′DelE4 PR4 Del 1-8 RHPHFPTRLQPPT V H W N G H E L M T P 34 326 2′Del C6 PR4 Del 1-8 RHPHFPTR LQPPT MG F M A H E L M V P 34 327 2′Del C11 PR4 Del 1-8 RHPHFPTR LQPPT A G L NE H E L L I P 34 328 2′Del D10 PR4 Del 1-8 RHPHFPTR LQPPT L A D N A R EL L T P 34 329 2′Del H5 PR4 Del 1-8 RHPHFPTR LQPPT L G K D V R E L L T P34 330 3′A7 PR4 Del 1-8 RHPHFPTR LQPPT L S D S G H A L F T P 34 3312′Del E3 PR4 Del 1-8 RHPHFPTR LQPPT L G P Y E H E L L T P 33 13 K10 Del1-8 RHPHFPTR LQPPT T G P N D R L L F V P 33 0.57 332 2′Del B5 PR4 Del1-8 RHPHFPTR LQPPT A G R H D H E L I I P 33 333 3′C12 PR4 Del 1-8RHPHFPTR LQPPT I G P N N H E L L T P 33 334 2′Del G9 PR4 Del 1-8RHPHFPTR LQPPT V E Q N G R E L I I P 33 335 2′Del C1 PR4 Del 1-8RHPHFPTR LQPPT A G L D E H E L L I P 32 336 3′E1 PR4 Del 1-8 RHPHFPTRLQPPT V A P N G H E L F T P 32 337 3′C3 PR4 Del 1-8 RHPHFPTR LQPPT M A QN G H A L F T P 32 338 2′DelB7 PR4 Del 1-8 RHPHFPTR LQPPT V G Y N N R EL L T P 32 339 3′F1 PR4 Del 1-8 RHPHFPTR LQPPT V A Q D G H F L Y T P 31340 2′Del B4 PR4 Del 1-8 RHPHFPTR LQPPT S G H N G H E V M T P 31 3413′G3 PR4 Del 1-8 RHPHFPTR LQPPT F D Q S D H E L L T P 31 342 2′DelH4 PR4Del 1-8 RHPHFPTR LQPPT V G P N E R M L M T P 30 343 3′D9 PR4 Del 1-8RHPHFPTR LQPPT G Y Y N D R E L L T P 30 344 3′G10 PR4 Del 1-8 RHPHFPTRLQPPT L T H N D H E L L T P 30 345 3′B2 PR4 Del 1-8 RHPHFPTR LQPPT V G RN D R E L L T P 29 346 2′DelC3 PR4 Del 1-8 RHPHFPTR LQPPT W A Q N G R EL L T P 29 347 3′F2 PR4 Del 1-8 RHPHFPTR LQPPT L G K N D H E L L T P 29348 4′C9 PR4 Del 1-8 RHPHFPTR LQPPT L G P N D H E L M T P 29 349 2′DelB2 PR4 Del 1-8 RHPHFPTR LQPPT T G W N G N E L F T P 29 14 K12 Del 1-8RHPHFPTR LQPPT D V Y N D H E I K T P 29 0.62 350 4′H7 PR4 Del 1-8RHPHFPTR LQPPT L A H N D H E L L T P 29 351 2′Del D1 PR4 Del 1-8RHPHFPTR LQPPT L E Q N D R V L L T P 28 352 2′Del H6 PR4 Del 1-8RHPHFPTR LQPPT T G H H D H E L I I P 28 353 3′B12 PR4 Del 1-8 RHPHFPTRLQPPT V A H E N R E L L T P 28 354 4′C5 PR4 Del 1-8 RHPHFPTR LQPPT L G LN D H E L I T P 27 15 K6 Del 1-8 RHPHFPTR LQPPT D G K D G R V L L T P 270.93 355 3′D8 PR4 Del 1-8 RHPHFPTR LQPPT A G P N D H Q L F T P 27 3563′C5 PR4 Del 1-8 RHPHFPTR LQPPT D A M Y G R E L M T P 27 357 3′A8 PR4Del 1-8 RHPHFPTR LQPPT V A W D D H E L L T P 27 358 2′Del F11 PR4 Del1-8 RHPHFPTR LQPPT M G Q N D K E L I T P 27 359 4′D8 PR4 Del 1-8RHPHFPTR LQPPT L A Q N G H E L Y T P 26 360 2′Del C5 PR4 Del 1-8RHPHFPTR LQPPT P G H N D H E L M V P 26 16 K15 Del 1-8 RHPHFPTR LQPPT EV H H D R E I K T P 25 0.35 361 3′B1 PR4 Del 1-8 RHPHFPTR LQPPT E A R NG R E L L T P 25 362 3′A9 PR4 Del 1-8 RHPHFPTR LQPPT L A H N D R E L L TP 25 363 4′B11 PR4 Del 1-8 RHPHFPTR LQPPT M A H N D H E L L T P 25 17K11 Del 1-8 RHPHFPTR LQPPT Q A P N D R V L Y T P 24 1.16 364 3D12 PR3Del 1-8 RHPHFPTR LQPPT L G Q N D R Q L L V P 24 365 2′Del H12 PR4 Del1-8 RHPHFPTR LQPPT A G G N G H E L L T P 24 366 3′H9 PR4 Del 1-8RHPHFPTR LQPPT H G P Y D Q V L L T P 24 367 3′F6 PR4 Del 1-8 RHPHFPTRLQPPT I E Q S G L Q L M T P 24 368 1′DelE6 PR4 Del 1-8 RHPHFPTR LQPPT LA Q N D R E L L T P 24 369 3′E5 PR4 Del 1-8 RHPHFPTR LQPPT V A W D G R EL F T P 23 370 3A3 PR3 Del 1-8 RHPHFPTR LQPPT L A Y N G R E I I T P 23371 3A2 PR3 Del 1-8 RHPHFPTR LQPPT W S Q N N R E L F T P 23 372 3′B11PR4 Del 1-8 RHPHFPTR LQPPT E T W N D H E I R T P 23 373 1′DelA2 PR4 Del1-8 RHPHFPTR LQPPT V A Q N G H Q L F T P 23 374 2′D6-PR4 WT RHPHFPTRLQPPT V T H N G H P L M T P 22 375 3′H1 PR4 Del 1-8 RHPHFPTR LQPPT F A QN D H Q L F T P 22 376 2′Del G11 PR4 Del 1-8 RHPHFPTR LQPPT G G Q M N RV L M T P 22 377 2′Del F5 PR4 Del 1-8 RHPHFPTR LQPPT L V H N D R E L L TP 22 378 1′DelE7 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N G H E L F T P 22 3792′E4-PR4 WT RHPHFPTR LQPPT V H W N G H E L M T P 22 380 2′Del F6 PR4 Del1-8 RHPHFPTR LQPPT L G W N D H E L Y I P 22 381 3′E10 PR4 Del 1-8RHPHFPTR LQPPT A G H K D Q E L L T P 21 382 4′A9 PR4 Del 1-8 RHPHFPTRLQPPT L A Q N N H E L L T P 21 383 4′G12 PR4 Del 1-8 RHPHFPTR LQPPT V AW N D H E I Y T P 21 384 3′B10 PR4 Del 1-8 RHPHFPTR LQPPT L A Q T G R EL L T P 21 385 2′DelH9 PR4 Del 1-8 RHPHFPTR LQPPT V G W S G H E L F V P20 386 3′H8 PR4 Del 1-8 RHPHFPTR LQPPT V G H N D R E L I T P 20 3872′DelA5 PR4 Del 1-8 RHPHFPTR LQPPT W N Q N G Q E L F T P 20 388 3B5 PR3Del 1-8 RHPHFPTR LQPPT F G Q N G H A L L T P 20 389 3C7 PR3 Del 1-8RHPHFPTR LQPPT R G L N D G E L L T P 20 390 3G2 PR3 Del 1-8 RHPHFPTRLQPPT F G P S D H V L L T P 20 18 K14 Del 1-8 RHPHFPTR LQPPT R E E N D HE L L I P 20 0.57 391 4′B12 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N N H E L LT P 20 392 4′B8 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N D H K L F I P 20 3932′Del F1 PR4 Del 1-8 RHPHFPTR LQPPT R D Q Y E H E L L T P 20 394 3′G1PR4 Del 1-8 RHPHFPTR LQPPT L A L N G H E L F T P 19 395 3′D2 PR4 Del 1-8RHPHFPTR LQPPT V E S N G H A L F V P 19 396 2′DelG5 PR4 Del 1-8 RHPHFPTRLQPPT V G Q N N H E L L T P 19 397 2′DelC7 PR4 Del 1-8 RHPHFPTR LQPPT WD Q N G H V L L T P 19 398 2′Del E5 PR4 Del 1-8 RHPHFPTR LQPPT E G L N DH E L I I P 19 399 3′C8 PR4 Del 1-8 RHPHFPTR LQPPT E G L N D H E L M I P19 400 3′G7 PR4 Del 1-8 RHPHFPTR LQPPT E G Q N D Q L L F I P 19 401 3′A6PR4 Del 1-8 RHPHFPTR LQPPT L A Q N G H E L L T P 19 402 4′G4 PR4 Del 1-8RHPHFPTR LQPPT V A Q N D R E L L T P 19 403 4′H5 PR4 Del 1-8 RHPHFPTRLQPPT L A Q N G H E L F T P 18 404 1′DelH12 PR4 Del 1-8 RHPHFPTR LQPPT VA Q N E R E L F T P 18 19 K8 Del 1-8 RHPHFPTR LQPPT V T H N G H P L M TP 18 3.3 405 2′Del D5 PR4 Del 1-8 RHPHFPTR LQPPT V A W N D H M L M T P18 406 3′F9 PR4 Del 1-8 RHPHFPTR LQPPT L G P N D R E L M T P 18 4072′H4-PR4 WT RHPHFPTR LQPPT V G P N E R M L M T P 17 408 4′H12 PR4 Del1-8 RHPHFPTR LQPPT V A H N D H E L L T P 17 409 1′DelD2 PR4 Del 1-8RHPHFPTR LQPPT V A K N D H E L L T P 17 410 4′E7 PR4 Del 1-8 RHPHFPTRLQPPT W A Q N D H E L L T P 17 411 1′DelH10 PR4 Del 1-8 RHPHFPTR LQPPT FA Q N D H E L L T P 17 20 K13 Del 1-8 RHPHFPTR LQPPT L A L K G H E L L TP 17 0.58 412 3C3 PR3 Del 1-8 RHPHFPTR LQPPT M E Q N G H E L F T P 17413 2′Del B3 PR4 Del 1-8 RHPHFPTR LQPPT D A P N G R E L M V P 17 4142′Del A2 PR4 Del 1-8 RHPHFPTR LQPPT G G R N G H T L L T P 17 415 3′F12PR4 Del 1-8 RHPHFPTR LQPPT L S Q T D H E L L T P 17 416 3B4 PR3 Del 1-8RHPHFPTR LQPPT V G Q N E H E L L T P 17 417 3F8 PR3 Del 1-8 RHPHFPTRLQPPT V A Q N G H E L K T P 17 418 1′DelH5 PR4 Del 1-8 RHPHFPTR LQPPT VA Q N D R E L F T P 17 419 1′DelD5 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N HH E L F T P 17 420 3′E11 PR4 Del 1-8 RHPHFPTR LQPPT V G P H D R E L L TP 17 421 2′C6-PR4 WT RHPHFPTR LQPPT M G F M A H E L M V P 16 422 4C9 PR3Del 1-8 RHPHFPTR LQPPT L A Q N D H E L L T P 16 423 3C9 PR3 Del 1-8RHPHFPTR LQPPT L V R N D H E L L T P 16 424 3F10 PR3 Del 1-8 RHPHFPTRLQPPT L A Q D D H E L L T P 16 425 2′Del A11 PR4 Del 1-8 RHPHFPTR LQPPTE D I R V L W L N T T 16 426 1′DelD1 PR4 Del 1-8 RHPHFPTR LQPPT V T Q ND H E L L T P 16 427 1′DelE2 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N D H E LL T P 16 428 1′DelF3 PR4 Del 1-8 RHPHFPTR LQPPT M A Q N D H K L F T P 16429 4′A5 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D H E L L T P 16 430 1′DelB8PR4 Del 1-8 RHPHFPTR LQPPT M A Q N D H E L L T P 16 431 4′B7 PR4 Del 1-8RHPHFPTR LQPPT V A Q N N H E L L T P 16 432 4F4 PR3 Del 1-8 RHPHFPTRLQPPT L A Q N D R E L I T P 15 433 4B11 PR3 Del 1-8 RHPHFPTR LQPPT V G QN N H E L I T P 15 434 3′G2 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N G H E L IT P 15 435 2′Del C8 PR4 Del 1-8 RHPHFPTR LQPPT T A H N G H E L L T P 15436 3′B8 PR4 Del 1-8 RHPHFPTR LQPPT L G Y H D H A L F T P 14 437 3′H10PR4 Del 1-8 RHPHFPTR LQPPT W A W N D H E L M T P 14 438 1′DelA1 PR4 Del1-8 RHPHFPTR LQPPT V A Q N D H E L L T P 14 439 4′D6 PR4 Del 1-8RHPHFPTR LQPPT M A Q N D H E L M T P 14 440 4F9 PR3 Del 1-8 RHPHFPTRLQPPT M A Q N D H E L L T P 14 441 4H5 PR3 Del 1-8 RHPHFPTR LQPPT V A QN G H E L I T P 14 442 2D12 PR3 WT RHPHFPTR LQPPT E G W I D H E I M I P14 443 3′F7 PR4 Del 1-8 RHPHFPTR LQPPT E G Q N G S E L I V P 14 444 4C11PR3 Del 1-8 RHPHFPTR LQPPT M A Q N D R E L I T P 14 445 4B6 PR3 Del 1-8RHPHFPTR LQPPT V G Q N D H E L F T P 14 446 1′DelE12 PR4 Del 1-8RHPHFPTR LQPPT V A Q S D H E L F T P 13 447 1′DelC2 PR4 Del 1-8 RHPHFPTRLQPPT V D R N D H E L F T P 13 448 1′DelA9 PR4 Del 1-8 RHPHFPTR LQPPT LA Q N D H E L M T P 13 449 1′DelA4 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N DH E L F T P 13 450 3G5 PR3 Del 1-8 RHPHFPTR LQPPT L G E N D R K L I T P13 451 4A12 PR3 Del 1-8 RHPHFPTR LQPPT V A Q N D H E L L T P 13 4522′Del E12 PR4 Del 1-8 RHPHFPTR LQPPT E G P N G H E L I T P 13 453 3G1PR3 Del 1-8 RHPHFPTR LQPPT M A Q N V R E L L T P 13 454 4F12 PR3 Del 1-8RHPHFPTR LQPPT V T Q N G H E L I T P 13 455 4B7 PR3 Del 1-8 RHPHFPTRLQPPT V T Q N D H E L F T P 13 456 4′G8 PR4 Del 1-8 RHPHFPTR LQPPT V A QN G H E L L T P 13 457 3′E8 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N D R Q L FT P 12 458 3′E4 PR4 Del 1-8 RHPHFPTR LQPPT V G P N D R E L I V P 12 4591′DelC6 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N E H E L L T P 12 460 1′DelD3PR4 Del 1-8 RHPHFPTR LQPPT L A Q N N H E L I T P 12 461 3A8 PR3 Del 1-8RHPHFPTR LQPPT E A H H G H E L M I P 12 462 3C5 PR3 Del 1-8 RHPHFPTRLQPPT G D H N D R E L M T P 12 463 2′G11-PR4 WT RHPHFPTR LQPPT G G Q M NR V L M T P 12 464 3′D4 PR4 Del 1-8 RHPHFPTR LQPPT L A H N D R E L I T P12 465 3E6 PR3 Del 1-8 RHPHFPTR LQPPT V P Q N G H E L I T M 12 4661′DelA11 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D H E L F T P 12 467 4′D12PR4 Del 1-8 RHPHFPTR LQPPT V D Q N D H E L F T P 12 468 2′D5-PR4 WTRHPHFPTR LQPPT V A W N D H M L M T P 11 469 2′A1-PR4 WT RHPHFPTR LQPPT SG H N D H M L M I P 11 470 1′DelG11 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N GH V L I T P 11 471 2′DelB10 PR4 Del 1-8 RHPHFPTR LQPPT V T H N D H E L LT P 11 472 2′DelB11 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N D H E L M T P 11473 1′DelC5 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D H E I M T P 11 474 4′B6PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D H E L I T P 11 475 3H9 PR3 Del 1-8RHPHFPTR LQPPT V S Q Q N H E L L T P 11 476 4E10 PR3 Del 1-8 RHPHFPTRLQPPT V A Q N D H E L M T P 11 477 3F5 PR3 Del 1-8 RHPHFPTR LQPPT V A YN E H E L Y T P 11 478 4A9 PR3 Del 1-8 RHPHFPTR LQPPT V A Q H D H E L LT P 11 479 1′DelH7 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N D Q E L L T P 11480 1′DelB10 PR4 Del 1-8 RHPHFPTR LQPPT V A R N D H E L M T P 11 4812′DelB9 PR4 Del 1-8 RHPHFPTR LQPPT V G P T D H E L L T P 11 482 3F11 PR3Del 1-8 RHPHFPTR LQPPT V G L T D H V L L T P 10 483 4C4 PR3 Del 1-8RHPHFPTR LQPPT V A Q D D H E L F T P 10 484 4B5 PR3 Del 1-8 RHPHFPTRLQPPT L A Q N D H E L F T P 10 485 3D4 PR3 Del 1-8 RHPHFPTR LQPPT V G WN D H E L I T P 10 486 4A4 PR3 Del 1-8 RHPHFPTR LQPPT V A Q N D H E L FT P 10 487 3D11 PR3 Del 1-8 RHPHFPTR LQPPT L G Q E N Q E L I T P 10 4882H10 PR3 WT RHPHFPTR LQPPT L A P S A R E L M T P 10 489 3G10 PR3 Del 1-8RHPHFPTR LQPPT V V H N G H E I L T P 10 490 3F4 PR3 Del 1-8 RHPHFPTRLQPPT M G Y E D H E L I T P 10 491 2H12 PR3 WT RHPHFPTR LQPPT E G Y Q NH E L S V P 10 492 4C2 PR3 Del 1-8 RHPHFPTR LQPPT V D Q N D H E L F T P10 493 1′DelG9 PR4 Del 1-8 RHPHFPTR LQPPT V A Q S D H E L M T P 10 4941′DelH9 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N D H E L I T P 10 495 1′DelB3PR4 Del 1-8 RHPHFPTR LQPPT V A Q N D H E L M T P 10 496 1′DelH1 PR4 Del1-8 RHPHFPTR LQPPT V A Q N G H E L I T P 9 497 3′A3 PR4 Del 1-8 RHPHFPTRLQPPT R A Q N D H E L I T P 9 498 1′DelC4 PR4 Del 1-8 RHPHFPTR LQPPT V AQ S N H E L M T P 9 499 1′DelE11 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N D RE L I T P 9 500 3F1 PR3 Del 1-8 RHPHFPTR LQPPT L T H N E Q Y L F T P 9501 2G9 PR3 WT RHPHFPTR LQPPT E I Y N D H E L M T P 9 502 3′D11 PR4 Del1-8 RHPHFPTR LQPPT M A Q N D H E L I T P 9 503 2′DelH2 PR4 Del 1-8RHPHFPTR LQPPT V S Q Y G H E L I T P 8 504 1′DelC10 PR4 Del 1-8 RHPHFPTRLQPPT V A K N D H E L I T P 8 505 4D2 PR3 Del 1-8 RHPHFPTR LQPPT V A Q NN H E L I T P 8 506 4A9 PR3 Del 1-8 RHPHFPTR LQPPT V A Q H D H E L L T P8 507 2F3 PR3 WT RHPHFPTR LQPPT L S H Y G K E L R T P 8 508 4A2 PR3 Del1-8 RHPHFPTR LQPPT V A Q N A H E L M T P 8 509 4G4 PR3 Del 1-8 RHPHFPTRLQPPT L G Q N D H E L L T P 8 510 1′DelB7 PR4 Del 1-8 RHPHFPTR LQPPT V AQ N D H E L K T P 8 511 3′D12 PR4 Del 1-8 RHPHFPTR LQPPT G E Q N D Y E LL V P 7 512 2′Del F12 PR4 Del 1-8 RHPHFPTR LQPPT L T Q H D H E L L T P 7513 4E2 PR3 Del 1-8 RHPHFPTR LQPPT M A Q N D H E L I T P 7 514 2C9 PR3WT RHPHFPTR LQPPT E A P N G R E L R T P 7 515 2′B9-PR4 WT RHPHFPTR LQPPTV G P T D H E L L T P 7 516 1′DelH6 PR4 Del 1-8 RHPHFPTR LQPPT V G Q Y DH E L I T P 6 517 4A3 PR3 Del 1-8 RHPHFPTR LQPPT V A Q D E H E L I T P 6518 2C12 PR3 WT RHPHFPTR LQPPT D A Q N V Q A P I A Q 6 519 2G12 PR3 WTRHPHFPTR LQPPT S G Q N D H A L L I P 6 520 2′A11-PR4 WT RHPHFPTR LQPPT ED I R V L W L N T T 6 521 2′C7-PR4 WT RHPHFPTR LQPPT W D Q N G H V L L TP 5 522 2′F7-PR4 WT RHPHFPTR LQPPT L G L R D R E L F V P 5 523 2C6 PR3WT RHPHFPTR LQPPT V E P N G H K L A I P 5 524 3′E6 PR4 Del 1-8 RHPHFPTRLQPPT F G Q N G K E F R I P 5 525 2′B4-PR4 WT RHPHFPTR LQPPT S G H N G HE V M T P 4 526 2′F6-PR4 WT RHPHFPTR LQPPT L G W N D H E L Y I P 4 5272′H5-PR4 WT RHPHFPTR LQPPT L G K D V R E L L T P 3 528 2F10 PR3 WTRHPHFPTR LQPPT L A L F D H E L L T P 3 21 VR28 WT RHPHFPTR LQPPT V A Q ND H E L I T P 3 11 22 159 WT RHPHFPTR LQPPA M A Q S G H E L F T P

TABLE 4 Sequences of characterized VEGF-R2 binding clones

TABLE 5 Affinities of the trinectin binders to KDR-Fc determined inradioactive equilibrium binding assay Clone KDR (Kd, nM) VR28 11.0 ± 0.5K1 <0.6 ± 0.1 K6 <0.9 ± 0.1 K9 <1.8 ± 0.4 K10 <0.6 ± 0.1 K12 <0.6 ± 0.1K13 <0.6 ± 0.1 K14 <0.6 ± 0.1 K15 <0.4 ± 0.1

TABLE 6 Affinities of the trinectin binders to KDR and Flk-1 determinedin radioactive equilibrium binding assay Clone KDR (Kd, nM) Flk-1 (Kd,nM) VR28 11.0 ± 0.5 nd* E3 <1.0 ± 0.2  5.4 ± 1.5 E5 <0.4 ± 0.1  3.2 ±0.3 E6 <0.4 ± 0.1  7.1 ± 1.1 E9  2.4 ± 0.3 <1.4 ± 0.1 E18 <1.2 ± 0.2<0.5 ± 0.1 E19 <1.3 ± 0.2 <0.7 ± 0.1 E25 <1.6 ± 0.4 <1.3 ± 0.2 E26 <1.7± 0.4  2.0 ± 0.3 E28 <1.6 ± 0.4  2.1 ± 0.6 E29 <1.5 ± 0.4 <0.9 ± 0.2*nd - binding is not detected at 100 nM of target

TABLE 7 Determination of ka, kd and Kd by BIAcore assay Clone Target ka(1/M*s) × 10⁻⁴ kd(1/s) × 10⁺⁵ Kd (nM) E6 KDR 89 6.7 0.08 Flk-1 67 136.02.02 E18 KDR 26 12.1 0.46 Flk-1 60 19.5 0.33 E19 KDR 30 1.7 0.06 Flk-166 22.3 0.34 E25 KDR 25 5.2 0.21 Flk-1 50 37.8 0.76 E26 KDR 11 5.8 0.51Flk-1 22 47.7 2.14 E29 KDR 36 7.0 0.19 Flk-1 79 28.8 0.37 M5FL KDR 109.2 0.89 Flk-1 28 58.2 2.10 VR28 KDR 3 34 13 159(Q8L) KDR 5 10 2

TABLE 8 Binding to KDR (CHO KDR) and Flk-1 (CHO Flk-1) expressing cellsCHO KDR CHO Flk-1 Clone (EC50, nM) (EC50, nM) E18 4.2 ± 1.0 0.9 ± 0.4E19 7.6 ± 1.7 5.3 ± 2.5 E26 2.6 ± 1.2 1.3 ± 0.7 E29 2.3 ± 1.0 0.6 ± 0.1WT no no

TABLE 9 Inhibition of VEGF-induced proliferation of KDR (Ba/F3-KDR) andFlk-1 (Ba/F3-Flk) expressing cells Ba/F3-KDR Ba/F3-Flk Clone (IC50, nM)(IC50, nM) E18 5.4 ± 1.2 2.4 ± 0.2 E19 12.3 ± 2.6  5.8 ± 1.0 E26 3.2 ±0.5 5.3 ± 1.7 E29 10.0 ± 2.1  4.7 ± 1.2 M5FL 3.9 ± 1.1 5.1 ± 0.2 WT nono Anti-KDR Ab 17.3 ± 7.7  ND Anti-Flk-1 Ab ND 15.0 ± 3.2 

TABLE 10 Inhibition of VEGF-induced proliferation of HUVEC cells Clone(IC50, nM) E18 12.8 ± 4.6 E19 11.8 ± 2.7 E26 14.0 ± 5.9 E29  8.4 ± 0.8M5FL  8.5 ± 2.8 WT no

TABLE 11 hKDR Flk-1 k_(a) k_(d) k_(a) k_(d) (1/Ms) × 10⁻⁴ (1/s) × 10⁵K_(D) (nM) (1/Ms) × 10⁻⁴ (1/s) × 10⁵ K_(D) (nM) M5FL C100 7.4 6.7 0.914.6 30 2.1 M5FL 20K 0.9 5.4 5.9 2.4 55 22.8 PEG M5FL 40K 0.5 5.9 1.31.0 54 57.1 PEG

1. A polypeptide comprising: a) a fibronectin type III (Fn3) domainhaving from about 80 to about 150 amino acids, wherein the Fn3 domainbinds to a target molecule with a K_(D) of less than 100 nM; and b) acovalently bound polyoxyalkylene moiety.
 2. The Fn3 polypeptide of claim1, wherein the Fn3 polypeptide comprises a ¹⁰Fn3 domain.
 3. The Fn3polypeptide of claim 2, wherein the Fn3 polypeptide comprises an aminoacid sequence that is at least 60% identical to SEQ NO:
 5. 4. The Fn3polypeptide of claim 3 wherein the Fn3 domain comprises, in order fromN-terminus to C-terminus, a) a beta strand or beta-like strand, A; b) aloop, AB; c) a beta strand, B; d) a loop, BC; e) a beta strand C; f) aloop CD; g) a beta strand D; h) a loop DE; i) a beta strand F; j) a loopFG; and k) a beta strand or beta-like strand, G.
 5. The Fn3 polypeptideof claim 4, wherein the loop FG participates in target molecule binding.6. The Fn3 polypeptide of claim 5, wherein the loops BC, DE and FGparticipate in target molecule binding.
 7. The Fn3 polypeptide of claim4, wherein each of the beta strands consists essentially of an aminoacid sequence that is at least 80% identical to the sequence of acorresponding beta strand of SEQ ID NO:
 5. 8. The Fn3 polypeptide ofclaim 4, wherein each of the loops AB, CD and EF consists essentially ofan amino acid sequence that is at least 80% identical to the sequence ofa corresponding loop of SEQ ID NO:
 5. 9. The Fn3 polypeptide of claim 1,wherein the polyoxyalkylene moiety is a polyethylene glycol (PEG)moiety.
 10. The Fn3 polypeptide of claim 3, wherein the polyoxyalkylenemoiety is a polyethylene glycol (PEG) moiety.
 11. The Fn3 polypeptide ofclaim 9, wherein the Fn3 polypeptide has a clearance rate of less than30 mL/hr/kg in a mammal.
 12. The Fn3 polypeptide of claim 11, whereinthe Fn3 polypeptide binds to the target molecule with a K_(D) of lessthan 10 nM.
 13. The Fn3 polypeptide of claim 2, wherein thepolyoxyalkylene moiety is attached to a thiol group or an amine group.14. The Fn3 polypeptide of claim 2, wherein the polyoxyalkylene moietyis attached to the Fn3 polypeptide by site directed conjugation.
 15. TheFn3 polypeptide of claim 13, wherein the polyoxyalkylene moiety isconjugated to a cysteine residue.
 16. The Fn3 polypeptide of claim 15,wherein the cysteine residue is not native to the Fn3 polypeptide. 17.The Fn3 polypeptide of claim 14, wherein the polyoxyalkylene moiety isconjugated to an amine.
 18. The Fn3 polypeptide of claim 13, wherein thepolyoxyalkylene moiety is conjugated to an ε-amino group of a lysineresidue.
 19. The Fn3 polypeptide of claim 18, wherein the lysine residueis not native to the Fn3 polypeptide.
 20. The Fn3 polypeptide of claim3, wherein the polyoxyalkylene moiety does not substantially decreasethe binding of the Fn3 polypeptide to the target molecule.
 21. The Fn3polypeptide of claim 20, wherein the polyoxyalkylene moiety is attachedat a position on the Fn3 polypeptide selected from the group consistinga) the N-terminus; b) between the N-terminus and the most N-terminalbeta strand or beta-like strand; c) a loop that does not participate intarget binding; d) between the C-terminus and the most C-terminal betastrand or beta-like strand; and e) at the C-terminus.
 22. The Fn3polypeptide of claim 9, wherein the PEG moiety has a molecular weight ofbetween about 2 kDa and about 100 kDa.
 23. The Fn3 polypeptide of claim22, wherein the PEG moiety is a branched PEG moiety.
 24. The Fn3polypeptide of claim 22, wherein the Fn3 polypeptide comprises a ¹⁰Fn3domain.
 25. The Fn3 polypeptide of claim 24, wherein the Fn3 polypeptidecomprises a ¹⁰Fn3 domain and has an amino acid sequence that is at least60% identical to SEQ NO:
 5. 26. A polypeptide comprising: a) afibronectin type III (Fn3) domain of about 80 to about 150 amino acids,wherein the Fn3 domain binds to a target molecule with a K_(D) of lessthan 100 nM; and, wherein the Fn3 domain comprises a cysteine residue ata position where a cysteine residue does not naturally occur, whereinthe position is selected from the group consisting of: a) theN-terminus; b) between the N-terminus and the most N-terminal betastrand or beta-like strand; c) a loop that does not contact the targetmolecule; d) between the C-terminus and the most C-terminal beta strandor beta-like strand; and e) at the C-terminus.
 27. The polypeptide ofclaim 26, wherein the cysteine is conjugated to an active moiety. 28.The polypeptide of claim 27, wherein the active moiety is selected fromthe group consisting of: a radioactive moiety, a chemotherapeutic agent,and a label.
 29. The polypeptide of claim 26, wherein the cysteine is asurface-exposed residue.
 30. The polypeptide of claim 26, wherein thepolypeptide binds to the target molecule with a K_(D) of less than 10nM.
 31. A polypeptide comprising a tenth fibronectin type III (¹⁰Fn3)domain of about 80 to about 150 amino acids and comprising a) a loop,BC; b) a loop, DE; and c) a loop FG; wherein, wherein the ¹⁰Fn3 domainbinds to a target molecule with a K_(D) of less than 100 nM, and whereinthe ¹⁰Fn3 domain comprises a cysteine residue at a position where acysteine residue does not naturally occur, wherein the position is notwithin loop BC, loop DE or loop FG.
 32. The polypeptide of claim 31,wherein the ¹⁰Fn3 domain comprises an amino acid sequence that is atleast 60% identical to SEQ NO:
 5. 33. The polypeptide of claim 31wherein the ¹⁰Fn3 domain comprises, in order from N-terminus toC-terminus, a) a beta strand or beta-like strand, A; b) a loop, AB; c) abeta strand, B; d) a loop, BC; e) a beta strand C; f) a loop CD; g) abeta strand D; h) a loop DE; i) a beta strand F; j) a loop FG; and k) abeta strand or beta-like strand, G.
 34. The polypeptide of claim 31,wherein the loop FG participates in target molecule binding.
 35. Thepolypeptide of claim 33, wherein each of the beta strands consistsessentially of an amino acid sequence that is identical to the sequenceof a corresponding beta strand of SEQ ID NO:
 5. 36. The Fn3 polypeptideof claim 33, wherein each of the loops AB, CD and EF consistsessentially of an amino acid sequence that is identical to the sequenceof a corresponding loop of SEQ ID NO:
 5. 37. The polypeptide of claim31, wherein the cysteine is conjugated to an active moiety.
 38. Thepolypeptide of claim 37, wherein the active moiety is selected from thegroup consisting of: a radioactive moiety, a chemotherapeutic agent, anda label.
 39. The polypeptide of claim 31, wherein the cysteine islocated in a loop that does not contact the target molecule.
 40. Thepolypeptide of claim 39, wherein the cysteine is a surface-exposedresidue.
 41. The polypeptide of claim 31, wherein the polypeptide bindsto target molecule with a K_(D) of less than 10 nM.
 42. A polypeptidecomprising a fibronectin type III (Fn3) domain of about 80 to about 150amino acids, wherein at least one loop of the Fn3 domain participates inbinding to a target molecule, wherein the Fn3 domain comprises anon-native lysine residue at a position selected from the groupconsisting of: a) the N-terminus; b) between the N-terminus and the mostN-terminal beta strand or beta-like strand; c) a loop that does notcontact the target molecule; d) between the C-terminus and the mostC-terminal beta strand or beta-like strand; and e) at the C-terminus.43. A polypeptide comprising: a ¹⁰Fn3 domain of about 80 to about 150amino acids and comprising a) a loop, BC; b) a loop, DE; and c) a loopFG; wherein at least one loop of the ¹⁰Fn3 domain participates inbinding to a target molecule, and wherein the ¹⁰Fn3 domain comprises anon-native lysine residue at a position that is not within loop BC, loopDE or loop FG.
 44. A pharmaceutical preparation comprising a polypeptideof claim
 1. 45. A pharmaceutical preparation comprising a polypeptide ofclaim
 26. 46. A pharmaceutical preparation comprising a polypeptide ofclaim 36.